Process for chemical manipulation of non-aqueous surrounded microdroplets

ABSTRACT

A process for the chemical manipulation of liquid and gel microdroplets is disclosed. The process involves providing first microdroplets having aqueous interiors, such that the first microdroplets are surrounded by a non-aqueous fluid. The aqueous interior chemical composition of the first microdroplets is then manipulated by exposure to compounds soluble in both the non-aqueous and aqueous phases, or by exposure to emulsions or suspensions of second microdroplets such that contact between the first and second microdroplets is made. This allows chemical manipulation to be accomplished without removing the first microdroplets from a non-aqueous fluid environment.

GOVERNMENT SUPPORT

The government has rights to this invention as it was sponsored in partby the National Institutes of Health by Grant RO1GM34077 and by the ArmyResearch Office by Contract Number DAAG29-85-K-0241 and by Grant NumberDAAG29-84-G-0066.

BACKGROUND OF THE INVENTION

Measurement, analysis and isolation of desirable cells is a wellestablished goal of much of industrial microbiology and biotechnology,with many important applications in fields such as clinicalmicrobiology, cancer diagnosis and treatment, environmental science,food safety, toxicology, and research and development in basic andapplied biology.

The importance of such isolation and prior methods for accomplishingisolation are well known (see, for example, Queener and Lively in Manualof Industrial Microbiology and Biotechnology, Demain and Solomon (Eds.),American Soc. Microbiol., Washington, D.C., 155-169, 1986; White et al,ibid, pp. 24-31). Selection strategies, which are based on preferentialgrowth and recovery of desired cells, are often possible. For example,selection can be accomplished by genetically manipulating cells suchthat a desired trait is coupled with resistance to antibiotics or to theability to grow in the absence of certain compounds. However, there aremany cases in which selection does not work well or does not work atall. In such instances, cells are often isolated by screening, i.e. bymethods wherein cells are cloned, identified and one or more desirableproperties measured, such that a particular clone is identified,physically removed, and thereby isolated.

One well established application of screening methods for microorganismsinvolves cells which have been exposed to mutagenic conditions, or togenetic splicing procedures, such that a large population of cellsresults, but of which only a very small number of cells are successes,e.g. cells which both grow and produce a desired molecule at a highlevel. Plating of such cells on petri dishes such that separatedcolonies arise is then followed by chemical assays. In such assays, asuccessful colony which is scored positive (+) by use ofradioimmunoassay, or is colored through use of a colorimetric indicator.The number of cells in such colonies is typically large. For example, inthe case of cells which are microorganisms such as bacteria, suchcolonies typically contain between 10⁷ and 10⁹ cells. Cells fromsuccessful colonies are then removed and suspended in a medium such thatthe number of cells can be enumerated, and the accumulated amount or theproduction rate of a desired molecule can be quantitatively determined.As is evident from this brief description, this type of procedurerequires several steps. Currently, the procedure is generally carriedout using relatively macroscopic entities and manual procedures, such aspetri dishes (for initial colony formation), visual inspection (foridentifying positive colonies), physical manipulation (for colonyremoval and suspension), physical measurement (for cell enumeration bylight scattering or other means) chemical assay (for determination ofmolecule concentrations at different times) and calculation (forcomparison of molecule production rate to known rates).

Screening is often much more difficult when a producing strain alreadyexists, and the task is to seek rare mutants which produce the samemolecule, but at higher rates, or to higher final concentrations. Insuch cases, the first step of scoring positive cells is unnecessary, butthen all cells must be quantitatively assayed for relative productivity.This is generally a very time consuming task. Because of the severalmacroscopic manipulations and assays, a typical screening task caninvolve formation of many initial colonies and significant manual labor.

The importance of measurement of biological entities, and the priormethods for accomplishing such measurement are also well known, withapplications in fields such as clinical microbiology, cancer diagnosisand treatment, environmental science, food safety, toxicology, andresearch and development in basic and applied biology. Measurementsrelating to biological entities such as cells, virus, nucleic acids andantibody-antigen complexes are of general interest. Although asignificant number of assays and tests are directed towards virus,nucleic acids and antibody-antigen complexes, the most well establishedmethods for determining measurements of such biological entities relatesto measurements of cells. For this reason, the main features of priormethods for the process for manipulation and measurement of non-aqueoussurrounded microdroplets are presented with an emphasis on prior methodsfor determining cell growth.

Present cell analysis methods involve two major classes of assays. Thefirst class rapidly detects and identifies specific cells directly froma primary sample, but does not determine cell viability. The most widelyused in this class are specific ligand binding assays, e.g. immunoassaysand genetic probes. However, they require many cells, and do notdistinguish between dead and viable cells. This restricts their use tosamples in which sufficient numbers of cells are present, and todeterminations in which direct assessment of the physiological state ofthe cell is irrelevant. The second class of assays is used for viablecell determinations either directly using the primary sample, or using asubculture of the primary sample. The most traditional and widely usedmethod is the plate count, which allows determination of single cellviability, based on growth, under many test conditions (see, forexample, Hattori The Viable Count: Quantitative and EnvironmentalAspects, Brock/Springer, Madison, 1988). An important attribute ofviable plate enumeration is that time required to obtain a determinationis independent of the concentration of the cell in the sample, becauseformation of each colony proceeds from an initial single cell. The majordisadvantage is its slowness, as typical determinations require one-halfto several days, and are also labor- and materials-intensive.

The disadvantages of viable plating can better be appreciated by drawingattention to its basic attributes. Viable plating is a well established,important method for qualitatively determining the growth of cells,particularly the presence or absence of growth for given conditions, andis often based on the growth of initial cells into distinct colonies.Viable plating typically involves the spreading of a suspension of cellsonto the surface of a gel-containing petri dish, with or without thepouring of a gel layer over the first gel surface. The gels are providedwith nutrients, such that following an incubation period at a suitabletemperature, many generations of growth occur. This leads to formationof visible colonies. For many microorganisms formation of visiblecolonies requires growth for 22 to 30 generations and therefore producescolonies containing 10⁷ to 10⁹ cells. (See Sharpe, in MechanizingMicrobiology, A. N. Sharpe and D. S. Clark (Eds.) Charles C. Thomas,Springfield, 19-40, 1978). Although conventional viable plating leads toformation of colonies, and thereby provides a basis for counting viablecells by counting colonies, the presence or absence of colonies onlyallows inference that the conditions present in the gel support do or donot support growth. For this reason, conventional viable plating is notwell suited to quantitative determinations such as cell growth rate andlag time, because viable plating based on visual inspection counts thenumber of colonies formed, but does not determine how the cellularmaterial or amount of cellular constituents in the colonies varies withtime. An additional complication arises because the nutrient andmetabolite concentrations within a colony comprise a microenvironment,which generally changes with time in an unknown way as microcoloniesincrease to form larger colonies with many cells in close proximity. Themicroenvironment within a large colony can also have significantheterogeneity of chemical composition within the microcolony, so thatdifferent cells within a large colony experience different growthconditions. Further, although some methods are based on astraightforward extension and application of scanning optical methodsfor determination of optical properties of colonies on or in gel slabs,such methods suffer from relatively large cost and size, and, because ofthe relatively large gel slab size, do not allow incubation conditionsto be changed rapidly at the site of the cells within the gel. (SeeGlaser in New Approaches to the Identification of MicroorganismsProceedings of a Symposium on Rapid Methods and Automation inMicrobiology, C.-G. Heden and T. Illeni (Eds.), Wiley, New York, 3-12,1975).

Instrumented methods for rapidly determining cell or culture growthand/or metabolic activity have been developed which only partiallyaddress the limitations of the viable plate assay. These include opticaltechniques for growth determination such as those which measure thechange in light scattering due to many cells in a liquid suspendedculture (See, for example, Edberg and Berger, in Rapid Methods andAutomation in Microbiology and Immunology, K. O. Habermehl, Ed.,SpringerVerlag, Berlin, 215-221, 1985), and a variety of metabolicactivity based techniques which measure changes due to many cells in ananalyzed sample. Examples of these include changes in extracellular pH(See, for example, Cowan and Steel's Manual for the Identification ofMedical Bacteria, Cambridge University Press, Cambridge, 1974; Manual ofMethods for General Bacteriology, P. Gerhardt, (Ed), American Societyfor Microbiology, Washington, 1981 ), carbon dioxide release (see, forexample, Courcol et al., J. Clin. Microbiol., 25: 26-29, 1986; Manca etal., J. Clin. Microbiol., 23: 401-403, 1986), electrical impedance (see,for example, Stewart, J. Exp. Med., 4: 235-245, 1899; Eden and EdenImpedance Microbiology, Research Studies Press, Letchworth, 1984; Hadleyand Yajko, in Instrumental Methods for Rapid Microbiological Analysis,Nelson (Ed.), VCH, Weinheim, 193-209, 1985; Bishop and White, J. FoodProtect, 49: 739-753, 1986), chemiluminescence (see, for example,Neufeld et al., in Instrumental Methods for Rapid MicrobiologicalAnalysis, Nelson (Ed.), VCH, Weinheim, 51-65, 1985; Rapid Methods andAutomation in Microbiology and Immunology, Habermehl (Ed.),Springer-Verlag, Berlin, 1985) or fluorescence (see, for example, Rossiand Warner in Instrumental Methods and Automation in Microbiology andImmunology, Habermehl (Ed.), Springer-Verlag, Berlin, 1985). Adisadvantage of all such metabolic activity methods is that they arebased on combined effects of a large number of cells. Therefore, thisgenerally requires an initial process, based on plating, to obtaininitial colonies for purposes of inoculation of the analyzed sample,such that the determinations based on many cells at least are based on amonopopulation, i.e. a population comprised norminally of the same typeof cells. For this reason, although a total population celldetermination may itself be rapid, it is generally preceeded by a viableplating method, or its equivalent, which is slow. Thus, the totalanalysis time, counted from receipt of a primary or non-plated sample toa cell growth determination, is the sum of both, and therefore stilllong.

Further, because such determinations are based on the combined effect ofa large, but unknown, number of cells, such total populationdeterminations do not actually yield a count. In contrast,determinations based on many individual measurements, each associatedwith an initial single cell, can yield a count.

Finally, because these total population methods are based on thecombined effects of many cells, the time required for a determinationbecomes significantly longer as the number of cells decreases, i.e. asthe sample's cell concentration decreases.

Similarly, prior use of flow cytometry for cell growth measurements(see, for example, Hadley et al. in Instrumental Methods for RapidMicrobiological Analysis, Nelson (Ed.), VCH, Weinheim, 67-89, 1985) islimited, because conventional use of flow cytometry performsmeasurements on individual cells, or clumps of cells which naturallyadhere, in an aqueous liquid suspension, and therefore does not have thecapability to measure colony formation. For this reason, prior use offlow cytometry can only measure total numbers of cells in a volume inorder to determine average growth, must also, therefore, involve acareful volume measurement, and is dependent on the signal-to-noiseratio of single cell measurements. This signal-to-noise ratio is lessthan satisfactory for many measurements (see, for example, ShapiroPractical Flow Cytometry, R. Liss, New York, 1985; Hadley et al. inInstrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.),VCH, Weinheim, 67-89, 1985).

Likewise, quantitative microscopy and image analysis combined withconventional gel preparations, such as gel slabs, petri dishes and thelike, although capable of determining colony formation, is tedious inmanual versions, and conventional gel slabs, petri dishes and the likecannot provide physical manipulability or a sufficiently fastcharacteristic diffusion time within the gel. Thus, cells cannot berapidly and conveniently exposed to different growth conditions, such asrapid changes in concentrations of nutrients, drugs, hormones, enzymes,antibodies and other chemicals. In addition, conventional gel slabs,petri dishes and the like cannot be readily manipulated physicallybecause of their size, and therefore cannot be readily used for exposureof gel-entrapped cells to in vivo conditions.

Previous studies of gel microdroplets have demonstrated that thesuspension of gel microdroplets (GMDs) in a non-aqueous fluid such asmineral oil results in retention of microbial metabolites such asmetabolic acids. This in turn allows detection and enumeration ofacid-producing microorganisms through the use of colorimetric andfluorescent pH indicators (Weaver et al., Ann. N.Y. Acad. Sci., 434:363-372, 1984; Williams et al, Ann. N.Y. Acad. Sci. 501: 350-353, 1987).In the previous work, observation of pH changes by optical means such ascolorimetry or fluorescence was accomplished using fluorescencemicroscopy. However, such prior use demonstrated that insignificantchemical communication of highly water soluble compounds, such asmetabolic acids, occurs between GMDs even when they are almostcontacting. Thus, such GMDs are found to be essentially chemicallyisolated, and cannot readily have the chemical composition of theaqueous interior of such GMDs altered or modified.

An attribute of these previous GMD methods is that GMDs surrounded bymineral oil (non-aqueous surrounded GMDs) were provide a chemicalisolation of GMDs, such that many chemicals are retained within GMDs.This retention within the very small volume of a GMD is the basis for animportant class of measurements on individual cells, individual enzymemolecules, microcolonies, and the like. In this previous use of GMDs,chemical retention is achieved by surrounding GMDs by a non-aqueousfluid which has solubility properties which essentially exclude thedissolution of many chemicals, particularly charged or ionic speciessuch as acids. For this reason, such chemicals do not significantlypartition into the non-aqueous fluid, and cannot therefore betransported through the non-aqueous fluid by a combination of diffusionand convection.

Although this is highly desirable in order to retain the extracellularproducts of biologically active entities within a GMD, and also toprovide or retain the chemical assay reactants used within a GMD as thebasis of an extracellular and/or intracellular assay, the previous usehas resulted in an inability to alter the chemical composition, forchemicals insoluble in the non-aqueous fluid, of the GMD interior oncethe GMDs were created and surrounded by a non-aqueous fluid.

It would, however, be highly desirable to be able to alter the chemicalcomposition of GMD interior fluids at one or more times after the GMDshave been surrounded by a non-aqueous fluid, under controlledconditions, and without the necessity of removing the GMDs from thenon-aqueous fluid followed by a re-suspension or re-surrounding of theGMDs by a non-aqueous fluid.

SUMMARY OF THE INVENTION

This invention pertains to a process for chemically manipulatingmicrodroplets which many contain biological entities, whereinmicrodroplets are surrounded by a non-aqueous fluid, such that bothwater soluble and ampiphillic species can be delivered to microdropletssurrounded by non-aqueous fluids, and additionally such that the amountof so delivered material can be subsequently determined for eachmicrodroplet. This process includes the use of emulsions containingliquid microdroplets and/or gel microdroplets, which can be cause tocollide with, and transfer chemicals to, other microdroplets.

DETAILED DESCRIPTION OF THE INVENTION Microdroplets

This invention relates to microdroplets, which are very small volumeentities comprised of liquid or gel material, and which can containzero, one or multiple biological entities. More specifically, the termmicrodroplet (MD) includes both the gel microdroplet (GMD), the liquidmicrodroplet (LMD), with or without contained biological entities. Thus,unless restricted by specific use of the term "gel microdroplet" or"liquid microdroplet", the term "microdroplet" refers to both gel andliquid microdroplets.

Liquid microdroplets (LMDs) are very small volumes of predominantlyliquid material, which can contain solutions, typically aqueoussolutions with inorganic and/or organic chemical compounds, and whichcan additionally contain biological entities. LMDs have volumes whichare defined by a boundary comprised of another liquid, such as anon-aqueous fluid, or by a permeability barrier such as a membrane, suchthat the membrane is capable of retaining biological entities ofinterest within a LMD, and also capable of passing other biologicalentities such as molecules.

Although LMDs can be of any shape, LMDs are often approximatelyspherical because of the tendency of interfacial forces associated withthe boundaries of LMDs to round up the deformable LMDs. Other forces,for example hydrodynamic shear associated with stirring a LMDsuspension, adhesion to a surface, or gravity, tend to cause departurefrom a spherical shape. Further, LMDs which contain or occupied byentities whose volume is a large fraction of the LMD volume can resultin LMDs which are non-spherical. Thus, for example, a cell surrounded bya thin coating of an aqueous solution, which in turn is surrounded by anon-aqueous fluid, is a LMD. Similarly, a non-biological particlesurrounded by a thin coating of an aqueous solution, which in turn issurrounded by a non-aqueous fluid, is also a LMD. If spherical, LMDshave diameters between about 0.2μ to about 1,000μ, preferably betweenabout 5μ and about 500μ. Generally LMD volumes are between about 8×10⁻¹⁵to about 1×10⁻³ ml, preferably between about 1×10⁻¹⁰ to about 1×10⁻⁴ ml.

Liquid microdroplets can be formed by a variety of methods, which aregenerally well known, and include methods based on breakup of a liquidjet, a spraying process, and by dispersion. For example, an aqueousliquid jet issuing into air can be forced to breakup into liquidmicrodroplets of nearly uniform volume (see, for example, Kachel andMenke in Flow Cytometry and Sorting, Melamed et al (Eds), Wiley, NewYork, pp. 41-59, 1979), or by spraying an aqueous liquid (see, forexample, Rotman, PNAS 47: 1981-1991, 1961). Likewise, the use ofdispersion to create emulsions consisting of a non-continuous aqueousphase of aqueous liquid microdroplets is well established (see, forexample, Weaver et al., Ann. N.Y. Acad. Sci., 434: 363-372, 1984).

GMDs are very small volume entities which comprise at least one gelregion, and which provide a mechanical matrix capable of entraping orsurrounding, without necessarily contacting, biological entities such assmall multicellular organisms, groups of cells, individual cells,protoplasts, vesicles, spores, organelles, parasites, viruses, nucleicacid molecules, antibody molecules, antigen molecules, and aggregates ofmolecules. GMDs can consist entirely of gel, in which case containmentof biological entities can occur by entrapment of the biologicalentities by the gel matrix. The general ability of gel matricies toentrap or immobilize biological entities is well known, having beenestablished for a variety of macroscopic gel preparations such as Petridishes, gel slabs and gel beads (see, for example, Immobilized Cells andOrganelles, Vols. I and II, Mattiasson (Ed), CRC, Boca Raton, 1983).

Alternatively, GMDs can consist of a shell of gel matrix material whichsurrounds at least one aqueous liquid region, in which case containmentof biological entites can occur by entrapment of the biological entitiesby the gel matrix material, or can occur by surrounding biologicalentities with a shell of gel matrix material, with or without contactingthe biological entities.

Further, GMDs can consist of a plurality of regions comprised of gelmaterial and liquid material. Representative configurations of GMDs witha plurality of gel regions include a first gel region entirelysurrounded by a second gel region, wherein the second gel region can becomprised of a gel material different from the gel material of the firstgel region. Alternatively, a second gel region can be comprise ofessentially the same gel material as a first gel region, but the secondgel can contain different entities such as entrapped beads andmacromolecules, or the second gel can have distinquishable moleculessuch as fluorescent molecules attached to a constituent of the secondgel matrix.

Similarly, GMDs can contain liquid regions which are surrounded by atleast one gel region. Representative GMDs with such liquid regionsinclude GMDs which consist of a shell of gel material which surrounds atleast one liquid region, such as an aqueous liquid core surrounded bygel. Such GMDs provide a general means for entrapping biologicalentities without necessarily contacting the biological entities with agel matrix, as it is only necessary that the gel matrix be impermeableto the surrounded biological entities, and that the gel matrix besufficiently mechanically strong that such GMDs remain intact during anydesired physical manipulation process of GMDs.

Liquid regions and gel regions of GMDs which contain no biologicalentities are termed nonbiological regions of a GMD.

Although GMDs can be of any shape, GMDs are often approximatelyspherical, with diameters between about 0.2μ to about 1,000μ, preferablybetween about 5μ and about 500μ. Generally GMD volumes are between about8×10⁻¹⁵ to about 1×10⁻³ ml, preferably between about 1×10⁻¹⁰ to about1×10⁻⁴ ml.

The term gel refers to a porous matrix with a high water content.Structures have the ability to entrap biological entities while allowingtransport of many molecules within the aqueous medium of the gel matrix.The gel matrix can also contain a chemical solution, typically anaqueous solution with inorganic and/or organic chemical compounds. Forexample, the gel matrix can contain a physiologic solution or cellgrowth medium, which is comprised of inorganic ions and molecules and/ororganic ions and molecules. Representative natural gel material forcreation of GMDs includes kappa-carrageenan, iota-carrageenan, sodiumalginate, furcelaran, zein, succinylated zein, succinlylated cellulose,agarose, collagan, fibrin, proteoglycans, elastin, hyaluronic acid andglycoproteins such as fibronectin and laminin, and other naturallyoccuring extracellular matricies, or the like. Representative syntheticgelable material synthetic water soluble polymers include those formedfrom vinyl pyrolidone, 2-methyl-5-vinyl pyrridine-methylacrylate-methacrylic acid copolymer, vinyl alcohol, vinyl pyrridine,vinyl pyrridine-styrene copolymer or the like.

GMDs can be created by a variety of methods, including the subdivisionof a macroscopic gel volume, but preferably GMDs are formed or createdby converting an aqueous suspension into liquid microdroplets, followedby formation of a gel state from the liquid state of the liquidmicrodroplets. Liquid microdroplets (LMDs) are very small, deformablevolumes of liquid which are surrounded by another distinct fluid, eitherliquid or gas, or are coated by a membrane material. A general processfor creating GMDs involves first creating LMDs, wherein the LMDs arecreated from a liquid which contains gelable material, such that uponsubsequent exposure to gelation conditions, the LMDs are transformedinto GMDs. Formation of the gel state can be caused by a variety of wellknown gelation processes, including temperature changes, ionconcentration changes, chemical concentrations, enzyme catalysis, andphotopolymerization.

The associated gelation processes may be reversible without hysteresis,reversible with hysteresis or irreversible. In the case of reversiblegelation without hysteresis, LMDs can be converted into GMDs, and GMDscan be converted into LMDs, by simply reversing the conditions, forexample returning to a temperature which first caused gelation. In thecase of reversible gelation with hysteresis, LMDs can be converted intoGMDs, and GMDs can be converted into LMDs, by reversing the conditionsbeyond the conditions needed to cause gelation, for example returning toand then passing a temperature which first caused gelation. In the caseof irreversible gelation, the conditions for reversing the gelationprocess cannot be achieved without creating conditions which are harmfulto the biological entities contained in the LMDs or GMDs. One example ofirreversible gelation is the formation of GMDs created byphotopolymerization.

In one general procedure the liquid suspension is forced through anozzle or vibrating orifice to form a liquid stream which breaks up,either because of the surface tension of a capillary jet, or byapplication of a shearing force, to form liquid microdroplets.Subsequently the liquid microdroplets are gelled by exposing the liquidmicrodroplets to conditions such as a temperature change, or bydirecting the liquid microdroplets into a solution containing ions whichcause gelation. One attribute of the nozzle or vibrating orifice GMDcreation method is that most GMDs are about the same size.

Another method for creating GMDs involves the first creation of amacroscopic gel volume, followed by subsequent fragmentation, cutting,disruption, or subdivision of the macroscopic gel volume such that aplurality of very small volume gel fragments or gel particles arecreated. This general method emphasizes that GMDs need not be spherical,nor even approximately spherical. Instead, it is only necessary thatGMDs consist of very small volumes of gel material, with volumes betweenabout 8×10⁻¹⁵ to about 1×10⁻³ ml, preferably between about 1×10⁻¹⁰ toabout 1×10⁻⁴ ml.

It is generally preferred to use a dispersion method for creating GMDsfrom a liquid suspension, as dispersion methods are simpler, lessexpensive and generally freeer from clogging problems than are fluid jetmethods. The dispersion methods consist of dispersing the liquidsuspension into an immiscible liquid such as a heavy alcohol, mineraloil or silicone fluid, by means such as stirring or vortexing the liquidsuspension and immiscible liquid together, thereby creating liquidmicrodroplets surrounded by the immiscible liquid. The liquidmicrodroplets are gelled during or after the dispersion process by anyof a variety of well known gelation processes such as ion exchange ortemperature change. Dispersion methods generally create GMDs with arelatively wide range of sizes, for example diameters of about 5μ to500μ.

GMDs can also be formed by the process of fragmenting, cutting,disrupting or otherwise converting a macroscopic volume of gel into verysmall volume gel particles, such that said GMDs can have irregularshapes. For example, a macroscopic gel slab can be formed in whichbiological entities such as cells are entrapped at random positions, thegel slab can be cooled to a low temperature, and the gel slab thenmechanically impacted so as to fragment the macroscopic gel into pieces,many of which have a very small volume and thereby constitute GMDs.

GMDs can also be formed by processes which cause a gel coating to formaround one or more entities, such that the gel entirely surrounds, oressentially surrounds, the entities. For example, by contacting coldcells with a warmer solution of material which contains gel materialwhich gels upon cooling, a coating of gel can be formed around thecells, such that the cells are thereby incorporated into GMDs. In thiscase the GMDs can be markedly non-spherical, as the gel coating oftenforms with the shape of the cells. Similarly, non-biological entitiessuch as cell culture microcarrier beads, soil particles and foodparticles can be incorporated into GMDs by gel-coating processes, suchthat the resulting GMDs often have shapes which approximate theincorporated non-biological entities.

In those cases wherein GMDs are formed within a non-aqueous fluid, it isoften desireable to transfer the GMDs into an aqueous medium, in orderto expose biological entities to a variety of conditions relating togrowth, metabolism, secretion, transmembrane potential development,membrane integrity and enzyme activity, and also to conditions whichfavor measurement and isolation of GMDs. An exemplary method for suchtransfer is gentle agitation if the GMD aqueous interiors containsuitable surfactant agents, including naturally occuring surfactantssuch as those present in serum.

Composite Gel Microdroplets

Composite GMDs are GMDs which contain more than one distinguishableregion of gel material or of liquid material, which gel or liquidmaterials are non-biological regions which may entrap or surroundbiological entities, and can be formed by several methods. Thus, acomposite GMD is characterized by a plurality of non-biological regionswhich are further characterized by having at least one non-biologicalregion having a first property surrounding substantially, or entirely,all of at least one non-biological region having a second propertyComposite GMDs can contain both gel and liquid regions, wherein liquidregions are surrounded by one or more gel regions, so that suchcomposite GMDs must contain at least one gel region. However, in orderto be useful for making measurements on biological entities, and forisolating biological entities, at least some composite GMDs containbiological material.

In one general method the aqueous suspending medium is provided with anycells, microbeads or other marker entities or force-coupling entitieswhich are desired to be incorporated or entrapped in a first gel region.First GMDs are then formed from a first gellable material, using any ofprocesses described elsewhere in this disclosure. The first GMDs arethen suspended in a medium containing a second gellable material, whichsecond gellable material may be of the same or different composition asthe first gellable material. Additionally, the second gelable materialcan be comprised, partially or entirely, of material which has opticalproperties such as light scattering, light absorbance or colorimetric,fluorescence, time-delayed fluorescence, phosphorescence andchemiluminescence, so as to distinguish the second gel region from thefirst gel region. In this way, by using any combination of one or moreof such methods, the second gel can provide composite GMDs, in this caseof two distinguishable gel regions, termed GMD/GMDs, with desirableoptical properties. For example, an optical signal for GMD diameterdetermination can be obtained from the second gel region while avoiding,with high probability, the contacting of first GMD entrapped cells withthe second gel region.

More generally, it is useful to form composite GMDs wherein at least oneregion contains a first material with a first optical property selectedfrom the group consisting of light scattering, light absorbance orcolorimetric, fluorescence, time-delayed fluorescence, phosphorescenceand chemiluminescence, and a second region contains a second, opticallydistinguishable material with optical properties selected from the groupconsisting of light scattering, light absorbance or colorimetric,fluorescence, time-delayed fluorescence, phosphorescence andchemiluminescence.

Alternatively, marker entities including beads, non-biologicalparticles, crystals, non-aqueous fluid inclusions, viable cells, deadcells, inactive cells, virus, spores, protoplasts, vesicles, stains anddyes can be incorporated into the first gel region or first GMDs, andbiological entities such as small multicellular organisms, groups ofcells, individual cells, protoplasts, vesicles, spores, organelles,parasites, viruses, nucleic acid molecules, antibody molecules, antigenmolecules, and aggregates of molecules can be incorporated into thesecond gel region in order to provide means for enhanced measurement ofcomposite GMDs. In this case, composite GMDs with at least one gelregion containing marker entities selected from the group consisting ofbeads, non-biological particles, crystals, non-aqueous fluid inclusions,viable cells, dead cells, inactive cells, virus, spores, protoplasts,vesicles, stains and dyes are provided Such composite GMDs areparticularly useful in the case that the optical properties of themarker entities are selected from the group consisting of lightscattering, light absorbance or colorimetric, fluorescence, time-delayedfluorescence, phosphorescence and chemiluminescence. Composite GMDs canalso be characterized by a plurality of non-biological regions which arefurther characterized by having at least one non-biological regionhaving marker entities surrounding substantially, or entirely, all of atleast one non-biological region having a second property.

In order to form composite GMDs, several general processes can be used.One method for producing GMDs having a plurality of non-biologicalregions, with at least one non-biological region having differentproperties than at least one other non-biological region, consists ofthe following general steps: (a) forming GMDs of a first gel using anyof the processes described elsewhere in this disclosure, (b) suspendingthe gel microdroplets in a material capable of forming a second gel, and(c) incorporating gel microdroplets of the first gel into gelmicrodroplets of the second gel, thereby forming gel microdroplets withdistinct non-biological gel regions.

Another general method for producing GMDs having a plurality ofnon-biological regions, wherein at least one non-biological region hasdifferent properties than at least one other non-biological region,consists of the following general steps: (a) forming GMDs, (b)suspending said GMDs in a material capable of forming LMDs, and (c)incorporating GMDs of the first gel into LMDs, thereby forming compositeGMDs with distinct non-biological regions, in this case composite GMDswith one or more liquid regions.

Still another general method for producing GMDs having a plurality ofnon-biological regions, wherein at least one non-biological region hasdifferent properties than at least one other non-biological region,involves the following general steps: (a) forming GMDs of a first gelcapable of liquification; (b) suspending said GMDs in a material capableof forming a second gel, (c) incorporating GMDs of said first gel intoGMDs of said second gel, and (d) liquifying the first gel, therebyforming GMDs with distinct non-biological liquid regions, in this casealso with at least one liquid region.

In order to use the gel microdroplets of this invention in processesinvolving measurement and/or isolation of biological entities, thecomposite GMDs should be formed from a suspension or solution whichcontains the appropriate biological entities, and therefore whichcontains biological material. The composite GMDs of this invention areuseful in cases wherein the biological material is composition ofbiological entities such as small multicellular organisms, groups ofcells, individual cells, protoplasts, vesicles, spores, organelles,parasites, viruses, nucleic acid molecules, antibody molecules, antigenmolecules, and aggregates of molecules, and is particularly useful incases wherein the cells are selected from the group consisting of animalcells, plant cells, insect cells, bacterial cells, yeast cells, fungicells and mold cells, or are selected from the group consisting ofnormal human cells, human cancer cells, pathogenic bacteria, pathogenicyeast, mycoplasms, parasites, and pathogenic viruses.

Force coupling entities such as beads, non-biological particles,bubbles, and non-aqueous fluid inclusions with force coupling propertiesselected from the group consisting of ferromagnetic properties,diamagnetic properties, paramagnetic properties, dielectric properties,electrical charge properties, electrophoresis properties, mass densityproperties, and optical pressure properties can be also incorporatedinto one or more gel regions, or liquid regions, in order to providemeans for physical manipulation of composite GMDs. Thus, it is useful toprovide composite GMDs which contain one or more regions with containforce-coupling entities such as beads, non-biological particles,bubbles, and non-aqueous fluid inclusions with force coupling propertiesSuch provision of force-coupling entities allows composite GMDs to bemanipulated by applying forces such as electrical force, magnetic force,field flow sedimentation fractionation force, acoustic force, opticalpressure force, gravitational force, sedimentation force, non-rotationalacceleration force, centrifugal force and centripetal force

The invention also includes extension of the basic process to thecreation of composite GMDs comprising more than two distinguishable gelregions. For example, composite GMDs which are GMD/GMDs can be used in aGMD formation process to form GMD/GMD/GMDs, that is, composite GMDs withthree distinguishable gel regions.

GMDs containing more than one non-biological gel region can be formed,such that composite GMDs are thereby formed, wherein said composite GMDsare comprised of regions of different gel material, and/or of the samegel material but with different entrapped or bound entities. Thus, forexample, a GMD which is a composite GMD made from two different gelmaterials can contain a first inner region which is comprised of a soft,low density gel such as 0.5% agarose, and a second outer region which iscomprised of a harder, higher density gel such as 4% agarose. Continuingthis illustration, the 0.5% agarose first inner region can support thegrowth of cells with less compressive force on the cells, while thesecond outer region can better confine the growing cells.

GMDs containing at least one liquid region can also be formed. Anexemplary process for formation of GMDs wherein a gel material regionsurrounds a liquid region is as follows. A first step comprises using aprocess to form GMDs from a gel material capable of subsequentliquification, a second step comprises formation of GMDs which consistof a second gel region completely surrounding the first GMDs, such thatthe second gel material is different from the first gel material, and iscapable of remaining a gel under conditions which liquify the first gel,and a third step comprises liquification of the first gel material, withthe result that GMDs with a liquid region surrounded by a gel region arethereby formed. A more specific illustration of this process is asfollows. The first step comprises forming liquid microdroplets whichcontain sodium alginate by forcing a suspension of biological entitieswith sodium aliginate through a vibrating orifice, thereby breaking upthe resulting liquid jet which contains both biological entities andsodium alginate, allowing the resulting liquid microdroplets to enter anaqueous medium containing calcium ions, thereby forming calcium alginateGMDs. The second step comprises concentrating the calcium alginate GMDsby means such as filtration and centrifugation, adding molten agarose atabout 37° C., following which the calcium alginate GMD suspension isdispersed into mineral oil, and cooling the dispersion, thereby formingagarose GMDs which contain alginate GMDs, The third step comprisesexposing said composite GMDs to an aqueous solution containing sodiumchloride and essentially zero calcium, such that sodium and calcium ionscan be exchanged, and thereby liquifying the calcium alginate within thecomposite GMDs.

For convenience it is useful to term GMDs comprised of more than one geland liquid region as composite GMDs, and to use notation such thatGMD/LMDs refers to GMDs formed with a first formed region which isliquid and a second formed region which is gel, and GMD/GMD refers toGMDs formed with a first region which is gel and a second formed regionwhich is gel. Thus, in the preceeding more specific illustration, uponcompletion of the second step GMDs comprising GMD(agarose)/GMDs(calciumalginate) are formed, and upon completion of the third step GMDscomprising GMD(agarose)/LMDs(sodium alginate) are formed.

Measurements of Microdroplets

The term measurement refers to the process of quantifying the amount ofa parameter, and includes the term detection, as detection is a coarsemeasurement which determines whether or not a parameter is greater thanor equal to a threshold condition, or is less than a thresholdcondition. Thus, for example, optical measurement of a microdropletinvolves quantifying at least one optical signal associated with amicrodroplet. The term quantifying refers to assigning a value to thesignal, such that said quantifying has a resolution which allows theparameter to be assigned one of two more than two different values. Incontrast, detection refers to measurement wherein the resolution allowsthe parameter to be assigned to only one of two values, one value whichcorresponds to subthreshold and therefore non-detection, and the otherwhich corresponds to threshold or suprathreshold and therefore todetection.

Although many useful processes relating to measurement and manipulationof microdroplets, and of any biological entities contained therein, canbe carried out without explicit measurements of microdroplet parameters,enhanced measurement and manipulation is often achieved if microdropletparameters such as microdroplet volume, V_(MD), microdroplet diameter,D_(MD), (if approximately spherical), microdroplet mass and microdropletmobility are determined.

The volume, V_(MD), of microdroplets can be measured by measuringsignals associated with the interface between two fluids which define aMD, or by signals associated with the difference in physical propertiesof the fluid within a MD and the fluid external to a MD. The physicalbasis for V_(MD) measurement can be selected from the group consistingof optical, weighing, sedimentation, field flow sedimentationfractionation, acoustic, magnetic, electrical and thermal measurement.

A variety of measurements can be based on a mass density differencebetween the fluid within a MD and the fluid external to a MD, andinclude weighing measurement on a microbalance such as a submergedpiezoelectric sensor, sedimentation measurement based on an accelerationfield such as gravity, rotational acceleration such as centripetalacceleration which is the basis of centrifugation, and/or non-rotationalacceleration, which is used to separate MDs on the basis of size andfield flow sedimentation fractionation measurement wherein MDs aregently separated according to size (see, for example, Levy and Fox,Biotech. Lab. 6:14-21, 1988). Other methods which are partially based ondifferences in mass density can also involve differences in otherparameters, and include measurements based on acoustic measurementwherein variation in acoustic properties of MDs relative to thesurrounding fluid are utilized (see, for example Quate, Physics Today,August 1985, pp. 34-42), magnetic measurement wherein differences inmagnetic properties, particularly paramagnetic, diamagnetic andferromagnetic properties, are utilized, and thermal measurement whereindifferences in thermal properties relative to the surrounding fluid areutilized, particularly differences in thermal conductivity, thermaldiffusivity and specific heat (see, for example, Bowman et al, Ann. Rev.Biophys. Bioengr. 4:43-80, 1975).

The generally preferred method of measuring V_(MD) involves opticalmeasurements selected from the group consisting of light scattering,light absorbance, fluorescence, phosphoresence and chemiluminescence, asoptical measurements are flexible, rapid and non-contactingmeasurements. Exemplary optical measurements using light scattering canbe based on differences in the index of refraction between the aqueousfluid within MDs and a non-aqueous fluid external to a MD, or can bebased on differences in light absorbance or colorimetry, phosphoresenceor chemiluminescence between the aqueous fluid within MDs and thenon-aqueous fluid.

More specifically still, it is preferred to utilize differences influorescence of the MD relative to the surrounding fluid, asfluorescence is particularly sensitive. Although both the fluid within aMD and external to MDs can be fluorescent, it is preferred to makemeasurements wherein either the fluid within the MD, or the non-aqueousfluid external to the MD, have significant fluorescence. Thus, forexample, at least one fluorescent molecule type can be incorporated inMDs, such that when surrounded by a non-fluorescent, non-aqueous fluidthe volume of a MD can be determined by the total fluorescence intensityassociated with the fluorescent molecule, and subject to the furthercondition that said fluorescent molecule not significantly partitioninto the surrounding non-aqueous fluid. Still more specifically, afluorescent molecule such as FITC-dextran can be incorporated into theaqueous medium which comprises the fluid within a MD, and the totalfluorescence emission intensity of FITC-dextran measured. Likewise, atleast one fluorescent molecule type can be incorporated into thenon-aqueous fluid which surrounds MDs, such that the decrease influorescence associated with the presence of a non-fluorescent MDprovides the basis of V_(MD) measurement (see, for example, Gray et al,Cytometry 3:428-434, 1983).

In addition to the measurement methods which can be used withmicrodroplets generally, GMDs containing one or more marker entities canalso be measured by measuring signals associated with at least onemarker entity which is incorporated into a gel matrix of at least oneGMD, wherein said marker entity is capable of measurement. In the caseof GMDs which exist prior to measurement, marker entities can beincorporated into the pre-existing GMDs prior to measurement.Alternatively, marker entities can be incorporated into GMDs bysupplying marker entities in the aqueous medium from which GMDs areformed, thereby incorporating marker entities into GMDs during theformation of GMDs

The volume, V_(GMD), of a GMD can be determined by first measuring themarker entities contained within a GMD, followed by analysis of theamount of marker entities in at least one gel microdroplet so as todetermine the size or volume of said gel microdroplet. Statisticalanalysis can be applied to one or more types of measurements relating toGMD properties, and/or to one or more types of measurements relating tobiological enities. It is preferred to combine measurement of biologicalentities with measurement of V_(GMD), so that statistical analysisrelating to the number of biological entities in GMDs can be used, andfor such combined measurements biological entities are incorporated intoGMDs prior to measurement of the GMDs based on marker entities.

Exemplary types of marker entities include beads, non-biologicalparticles, crystals, nonaqueous fluid inclusions, viable cells, deadcells, inactive cells, virus, spores, protoplasts, vesicles, stains anddyes. Non-biological particles include particles comprised of inorganicmaterial such as silica, of organic material such as charcoal or carbon,and of combinations of inorganic and organic material wherein theorganic material can be of biological or non-biological origin. Suchmarker entities allow a variety of measurement to be made, includingoptical, weighing, sedimentation, field flow sedimentationfractionation, acoustic, magnetic, electrical and thermal measurementsHowever, it is preferred to measure GMDs optically by using markerentities which can be measured by using light scattering, lightabsorbance or colorimetric, fluorescence, time-delayed fluorescence,phosphorescence and chemiluminescence.

In cases wherein the presence of marker entities does not adverselyaffect biological entities contained within GMDs, or the ability of gelmaterial to form a gel matrix, gellable material can be pretreated so asto attach at least one type of marker entity to at least one gellablematerial prior to formation of GMDs, thereby resulting in formation ofGMDs having enhanced measurement properties. Thus, for example,pretreatment of gelable material by chemically attaching marker entitiescomprising fluorescent molecules renders the GMDs by measurable byfluorescence. Alternatively, GMDs can be first formed, and markerentities subsequently introduced. For example, macromolecules such asdextrans can be labeled with a fluorescein derivative, agarose GMDsexposed to said macromolecules, whereupon the fluorescent dextran candiffuse into the agrose, and then be subsequently precipitated orcomplexed, such that the dextran is essentially trapped within the gel,and the GMDs are thereby provided with marker entities in the form offluorescent labeled dextran.

In some preparations of GMDs, a significant number of biologicalentities, such as cells, can either remain free in suspension, or canescape from some types of GMDs during vigorous physical manipulation.This results in suspensions which contain both cells trapped within GMDsand free cells, wherein the term free cells refers to cells free insuspension and not contained within MDs. This occurrance is usuallyundesirable. Thus it is highly desirable to provide measurement meansfor distinguishing free cells from cells contained within GMDs. Forexample, the formation of microcolonies within GMDs provides a generalmethod for determining growth of biological entities, particularlycells, and allows direct determination of plating efficiency followingan incubation by quantitatively comparing the number of microcolonies tosingle cells. However, if significant free cells can sometimes occur insuspension, and free cells usually cannot be distinguished from cellscontained within GMDs, so that significant error can result. In suchcases it is preferred to measure at least one type of marker entitycontained within GMDs, such that it is possible, by measurement of bothmarker entities and biological entities, to determine that there is ahigh probability that the biological entity is associated with a gelmicrodroplet. In other cases the measurement of marker entities cancomprise detection, wherein the detection of one or more GMDs allowsmeasurements of biological entities to be associated, with highprobability, with the containment of said biological entities withinGMDs. For example, non-growing individual cells within GMDs can bedistinguished from individual cells which are free in suspension.

Often it is also desirable, in order to enhance subsequent statisticalanalysis, to determine V_(GMD) for a GMD associated with a measuredbiological entity. In this case the measured parameter used for a markerentity type is selected to be distinguishable from measured parameterswhich relate to measurements of biological entities. For example,biological entities which contain double stranded nucleic acids can bemeasured by using well known staining protocols utilizing propidiumiodide (PI), and measuring the Red Fluorescence associated with PI,while marker entities such as entrapped microbeads, or covalentlyattached fluorescein, with Green Fluorescence provide the basis formeasurement of V_(GMD), as the magnitude of the Green Fluorescencesignal is proportional to V_(GMD). The combined measurements of V_(GMD)and bilogical entities further provide the basis for determining thefrequency-of-occupation of GMDs by biological entities, and therebyenhance statistical analysis methods such as those provided by usingPoisson statistics or modified Poisson statistics.

Marker entities can be selected with a variety of physical propertieswhich result in enhancement of GMD measurement upon incorporation ofsaid marker entities into GMDs. Useful physical properties which providethe basis for measurement of marker entities include optical properties,mass density properties, acoustic properties, magnetic properties,electrical properties and thermal properties. Because of their speed,specificity, non-perturbing nature and non-contacting nature, it ispreferred to use optical measurement means, including flow cytometryapparatus, flow-through-microfluorimetry apparatus, optical particleanalyzers apparatus, fluorescence microscopy apparatus, light microscopyapparatus, image analysis apparatus and video recording apparatus tomeasure marker entities. More specifically, in the case of flowcytometry, it is useful to measure optical pulses such as maximum pulsemagnitude, pulse time integral and pulse duration, all of which are wellknown (see, for example, Shapiro, Practical Flow Cytometry, A. R. Liss,New York, 1985).

The marker entities can also be measured using well known electricalmeasurements, particularly electrical resistance measurements,electrical measurements which provide the basis of particle analysis,such as the electrical resistance based particle measurements (see, forexample, Kachel in Flow Cytometry and Sorting, Melamed et al (Eds),Wiley, New York, pp. 61-104), and dielectric property measurement (see,for example, Harris et al, Enzyme Microb. Technol. 9: 181-186, 1987).These electrical measurements are well known and generally desirablebecause of the relative ease and relative low cost of making suchelectrical measurements.

Statistical Analysis of Measurements

The use of microdroplets generally provides means for making a largenumber of individual measurements relating to a biological sample. Thisis in constrast to most established measurement methods, as mostestablished measurement methods are responsive to the total effect onmeasured parameters by biological entities contained in a sample. Thus,although useful measurements can be made using small numbers ofmicrodroplets, in general, the use of large numbers of individualmicrodroplet measurements provides the basis for making significantlyimproved measurements on biological entities of a sample. Althoughsignificant measurement information can be obtained without explicitlycarrying out statistical analysis of large numbers of microdropletmeasurements, significant improvement in measurements is achieved byapplying statistical analysis to microdroplet measurements.

Measurements on microdroplets are often made wherein more than oneparameter is measured. For example, it is generally preferred to useoptical measurements, particularly fluorescence measurements, in whichcase simultaneous measurements such as Green Fluorescence measurementand Red Fluorescence measurement are often made. In the exemplary caseof measurements relating to a mixed biological population, a GreenFluorescence labeled antibody can be used to measure the amount ofbiological material associated with a first type of biological entity,and a Red Fluorescence labeled antibody can be used to measure theamount of biological material associated with a second type ofbiological entity. Following an incubation the magnitude of the GreenFluorescence and Red Fluorescence signals can be used to determine theamount of growth of each type of biological entity, such that thefrequency-of-occurrence distribution of the Green and Red Fluorescencesignals can be obtained, and then statistically analyzed to determinethe variation in growth, and the variation in lag time, for both typesof biological entities. In this exemplary case, however, it is notnecessary to use statistical analysis in combination with microdropletvolume measurement, but only with the Green and Red Fluorescencemeasurements. Thus, although statistical analysis of microdropletmeasurements is generally useful, statistical analysis does notnecessarily involve the use of microdroplet volume measurements, nordoes statistical analysis necessarily relate to occupation ofmicrodroplets. Instead, statistical analysis can relate to thefrequency-of-occurence of measurements relating to the biologicalentities themselves.

Individual and Multiple Microdroplet Occupation

In many cases it is useful to determine, at least approximately, thestatistical distribution of occupation of microdroplets by biologicalentities. As used herein, the term "occupation" refers to the presenceof initial biological entities, that is, those biological entitiespresent shortly after formation of microdroplets, and before anyincubation is used. Thus, for example, in the representative casewherein biological entities are cells, microdroplets can have a highprobability of zero occupation, individual occupation, or of multipleoccupation. As used herein, zero occupation or unoccupied refers to thecase wherein a microdroplet contains zero initial cell, individualoccupation refers to the case wherein a microdroplet contains oneinitial cell, and multiple occupation refers to the case wherein amicrodroplet contains at least two initial cells. Following anincubation, growth may occur and result in increases in size and numberof cells, such that an individually occupied microdroplet subsequentlycontains progeny cells of the initial single cell, and is neverthelesstermed an individually occupied MD, and a multiply occupied microdropletsubsequently contains progeny cells of the initial multiple cells, andis nevertheless termed a multiply occupied MD.

In the general case wherein a microdroplet contains at least two typesof biological entities, the term occupation can be used separately witheach type of biological entity. Thus, for example, in the case of amixed biological population comprised of two types of cells, type A andtype B, a microdroplet can initially contain, prior to any incubation,one A cell and several B cells. In this case the microdroplet is termedindividually occupied by type A cells and multiply occupied by type Bcells, and the same designation is also used subsequent to anyincubation which results in growth. That is, continuing this example, ifincubation subsequently leads to A cell progeny, the microdrop is stilldeemed individually occupied by type A cells. This terminology isstraightforwardly extended to all types of biological entities.

Analysis Using Poisson Statistical Methods

It is often preferred to make measurements on microdroplets that areindividually occupied, so that the measurements can be interpreted asmeasurements relating to one biological entity. In order to formmicrodroplets which have a significant fraction of microdroplets withindividual occupation, it is generally useful to estimate thedistribution of occupation for different size microdroplets. Manymethods for forming microdroplets, in which biological entities areincorporated into microdroplets, are random, or well approximated byrandomness, such that statistical analysis involving one or more MDparameters, such as diameter or volume, is useful for determining theprobability of occupation of different size microdroplets.

As a result, it is generally useful to determine the size ofmicrodroplets that have a high probability of having zero, individual ormultiple occupation, so that the probability of having less than twoinitial biological entities, and of having at least two initialbiological entities, in microdroplets of different size or volume rangescan be estimated. If the concentration of the suspended biologicalentities is known approximately, or can be estimated, then thesuspension can be diluted so as to provide an average, known number ofbiological entities in liquid microdroplets of a particular size orvolume being made. A mathematical formula or equation which describesthe relation between the average number of biological entities andliquid microdroplet volume is the Poisson probability distribution,P(n,n), (see, for example, Gosset, Biometrika, 5: 351-360, 1907; Weaveret al., Ann. N.Y. Acad. Sci., 434: 363-372, 1984; Weaver, Biotech. andBioengr. Symp. 17, 185-195, 1986; Williams et al., Ann. N.Y. Acad. Sci.,501: 350-353, 1987), which in its application to microdroplets gives theprobability, P(n,n), of finding a particular number, n, of initialbiological entities in microdroplet volume V_(MD) if the average numberof initial biological entities found in the volume V_(MD) is n. Morespecifically ##EQU1## A mathematical relation governing n is n=ρV_(MD),where ρ is the concentration of the biological entities in thesuspension which was converted into liquid microdroplets, and the term"average occupation" is defined to be n, and refers to the average ormean number of initial biological entities present before anyincubation. For a sample with randomly distributed biological entities,such as a well-stirred sample, the probability of finding particularnumbers of biological entities is described by the Poisson formula.Thus, the probabilty of having zero initial biological entities or beingunoccupied is P(0,n), the probability of having one biological entity orbeing individually occupied is P(1,n), the probability of having twobiological entities is P(2,n), and so on. Of particular interest to someapplications of GMDs is the situation wherein a LMD is initiallyoccupied by more than one entity, that is, multiply occupied. Theprobability of multiple occupation is P(>1,n), and, because the sum ofall possible probabilities equals one, the probability of initialmultiple occupation is as follows:

    P(>1,n)=1-P(0,n)-P(1,n)                                    (2)

Generally, the transition from LMDs to GMDs does not involve significantchanges in volume, i.e. V_(MD) =V_(LMD) ≈V_(GMD) in most cases. In suchcases the Poisson probability can be used interchangeably with eitherLMDs or GMDs. If the volume change is significant, a volume scalingfactor, f_(vol), is used according to V_(GMD) =f_(vol) V_(LMD), suchthat there is a common relation between the volumes of all GMDs and theLMDs from which they were created. This scaling factor, f_(vol), isgenerally a well known macroscopic property of gel materials.

In the process of creating MDs, a sample volume, V_(S), is mixed with avolume of additional material, V_(Add). In the case of GMD formation,the volume V_(Add) usually contains gellable material which forms thegel matrix of the subsequent GMDs. The corresponding dilution by afactor f_(D) is as follows: ##EQU2## and is straightforwardly computedfollowing measurement of both V_(S) and V_(G). The diluted biologicalentity concentration, ρ, is therefore related to the sample biologicalentity concentration, ρ_(S) by the following: ##EQU3## where ρ is thebiological entity concentration used in the Poisson equation.

In the case of biological entities which naturally aggregate, in thePoisson equation, the mathematical parameters n, n and ρ_(s) refer tothe particular number of aggregates, the average number of aggregatesand the concentration of aggregates, respectively. Thus, for biologicalentities which naturally aggregate as colony forming units (CFU), in thePoisson equation n, n and ρ_(s) refer to the particular number of CFUs,the average number of CFUs and the concentration of CFUs, respectively.

In some cases it is desirable to make measurements on more than one MDat a time, and thereby to make measurements on clusters or groups of MDswhich are associated with individual MDs. In this case the Poissonstatistics formula is applied with the change that V_(MD) is replaced byV_(group), wherein V_(group) is the sum of the individual MD volumes inthe group.

In some cases, such as those wherein the volume of n biologicalentities, nV_(BE), within a MD is a significant fraction of the volumeof a MD, V_(MD), it is useful to employ a modified form of the Poissonformula. An example of a modified Poisson function is given in equation(5). ##EQU4## in which the "available volume"=V_(MD) -nV_(BE) replacesV_(MD), which provides a better description of the frequency ofoccupation for cases wherein V_(BE) is a significant fraction of V_(MD).This approximate equation uses the definition that the volume of a MDcontaining n cells is defined to be the volume of the gel material plusnV_(BE), and is consistent with the inability of two or more nominallyidential biological entities initially occupying a MD of volume lessthan 2V_(BE). In the case that non-identical biological entities areinitially present, the quantity nV_(BE) is replaced by V_(total),BE, thetotal volume of the biological entities.

Another approximate, modified version of the Poisson function is is mostreadily presented in terms of a separate mathematical expression foreach of the probabilities, P(0,n), P(1,n), P(2,n), etc. The first twoexpressions are

    P(0,n)≈1 if V.sub.MD <V.sub.BE and e.sup.(-n+ρV.sbsp.BE) if V.sub.MD ≧V.sub.BE                                 (6a)

    P(1,n)≈0 if V.sub.MD <V.sub.BD ; (1-e.sup.(-n+ρV.sbsp.BE) if V.sub.BE <V.sub.MD ≦8V.sub.BE                      (6b)

These modified Poisson forumulae or equations agree with equation (1) inthe mathematical limit that V_(BE) approaches zero, but provide betterdescriptions results in cases wherein the volume of the initialbiological entities within a MD is a significant fraction of V_(MD).

Further, other extensions or modifications of statistical analysis canbe used to analyze cases wherein the analyzed MDs have a significantrange of volumes. The basic concept underlying such analysis is thateach size range of MDs separately obeys the same probability equation,specifically Poisson statistics or modified Poisson statistics.

The preferred embodiment of the invention initially uses conventionalPoisson statistics for all cases, but then uses either the version givenby equation (5) or by equations (6a), (6b), (6c) and extensions thereof.if occupations of small volume MDs differ significantly from thoseobtained from large volume MDs in the same preparation made from thesame sample. It is also possible to utilize any other extension ofPoisson statistics which provides descriptions of the probability ofoccupation in the case that V_(BE) is a significant fraction of V_(MD).Depending on the relative size, shape and means for forming MDs, otherversion of modified Poisson statistics formulae may be utilized.

The most conceptually simple measurements relate to measurements whichsimultaneously measure one MD. Such measurements can be made using avariety of measurement apparatus based on optical, weighing,sedimentation, field flow sedimentation fractionation, acoustic,magnetic, electrical and thermal means, but preferably optical means inthe form of flow cytometry apparatus, flow-through-microfluorimetryapparatus, optical particle analyzers apparatus, fluorescence microscopyapparatus, light microscopy apparatus, image analysis apparatus andvideo recording apparatus. Electrical means can include dielectricproperty measurement apparatus or a particle analyzer based onelectrical resistance measurement As before, microdroplet measurementsare made in such measurement apparatus by operating the apparatus in amode wherein there is a high probability that less than two MDs aresimultaneously within the measurement volume of the apparatus. Thus, inthe exemplary case of flow cytometry, the instrument is operated in amode wherein there is a high probability that less than two MDs aresimultaneously in the focal volume of the optical illumination region.Further, in the exemplary case of microscopy, the instrument is operatedin a mode wherein there is a high probability that less than two MDs aresimultaneously in the field view used in a measurement. A generaladvantage relating to making measurements on less than two MDssimultaneously relates to simplicity of interpretation, as measurementscan be interpreted in terms of measurements on individual biologicalentities or their progeny.

Somewhat more conceptually complex measurements relate to measurementswhich simultaneously measure at least two MDs, wherein a plurality ofMDs is termed herein as a group or cluster of MDs. As describedelsewhere in the present disclosure, such measurements can be made usinga variety of measurement apparatus based on optical, weighing,sedimentation, field flow sedimentation fractionation, acoustic,magnetic, electrical and thermal means, but preferably optical means inthe form of flow cytometry apparatus, flow-through-microfluorimetryapparatus, optical particle analyzers apparatus, fluorescence microscopyapparatus, light microscopy apparatus, image analysis apparatus andvideo recording apparatus, or electrical means in the form of dielectricproperty measurement apparatus or a particle analyzer based onelectrical resistance measurement Microdroplet measurements are made insuch measurement apparatus by operating the apparatus in a mode whereinthere is a high probability that at least two MDs are simultaneouslywithin the measurement volume of the apparatus. Thus, in the exemplarycase of flow cytometry, the instrument is operated in a mode whereinthere is a high probability that less than two MDs are simultaneously inthe focal volume of the optical illumination region. Further, in theexemplary case of microscopy, the instrument is operated in a modewherein there is a high probability that less than two MDs aresimultaneously in the field view used in a measurement. Advantagesrelating to making measurements on at least two MDs simultaneouslyinclude larger measurement throughput rates of MDs, for example in caseswherein many MDs are unoccupied, and reduced technical complexity ofmeasurement apparatus because of generally larger measurement regionvolume.

The most conceptually simple use of microdroplets involves MDs withindividual occupation, as measurements of individually occupied MDs canbe readily related to measurements of biological material associatedwith an initial individual biological entity. For example, measurementof an individually occupied MD provides the basis for analysis andinterpretation of growth of an initial biological entity.

A more conceptually complex use of microdroplets involves microdropletswith multiple occupation. Multiple occupation may be selected for avariety of reasons, such as allowing more biological entities to bemeasured for the same number of MDs, or to provide biological entitiesinitially at a higher concentration within MDs. The multiply-occupied MDmeasurement itself, however, is essentially equivalent to themeasurement of multiple MDs, that is, measurement of groups or clustersof two or more MDs. As used herein, measurement of multiple MDs consistsof either the making or combining of measurement of two or more MDs. Forexample, the multiple measurement of five MDs consists either of makinga simultaneous measurement on five MDs, or on making measurements of anysubgroup of the five MDs followed by summing the subgroup measurementsto obtain the multiple MD measurement.

Even though measurement results cannot be as readily interpreted interms of individual biological entities, advantages of makingmeasurements on multiply occupied MDs include making measurements onbiological entities at a higher rate in comparision to individuallyoccupied MDs, and to lower costs associated with requiring less materialand time to make measurements. In addition, important information canoften be obtained which cannot be readily obtained from nonmicrodropletmeasurement methods. Specifically, measurement of multiply occupied MDsprovides the basis for analysis and interpretation of growth of initialbiological entities, wherein said growth is the average growthassociated with the occupation of the MD. Similarly, secretion by onehyperactive biological entity, or its progeny, can be measured in thepresence of other, poorly secreting biological entities. For example, inthe case that MDs with average occupation by three cells is measured,the measurement can still reflect any significant variability which ispresent in the cells, as the measurement of average property of threecells can be significantly affected by one unusual cell, therebydetermining that an unusual cell is present. Measurements can be usefulwith multiply occupied MDs containing 2 to about 10³ initial biologicalentities, but are preferably used with multiply occupied MDs containing2 to about 10 initial biological entities.

For example, consider the illustrative case wherein according to Poissonstatistics the mean or average occupation is three, so that n=3, themathematical Poisson probability formula predicts the distribution ofparticular occupation values, n, given in the table below.

    ______________________________________                                        n      P(n,- n = 3)    n     P(n,- n = 3)                                     ______________________________________                                        0      0.050           5     0.101                                            1      0.149           6     0.050                                            2      0.224           7     0.020                                            3      0.224           8     0.008                                            4      0.168                                                                  ______________________________________                                    

In this illustration, there is a low probability (0.05) of havingunoccupied MDs, a high probability (0.95) of having occupied MDs. Theprobability of individual occupation is 0.15 so the probability ofmultiple occupation is 0.80. Thus, 80% of MDs initially contain at leasttwo biological entities. However, even in this example for which themean occupation is high, the multiply occupied MDs have a peakeddistribution of occupation, such that there are equal, maximumprobabilities of 0.224 of having either 2 or 3 initial biologicalentities, and rapidly decreasing probabilities of having 4 or moreinitial biological entities. As a result, the probability, P(>7,n) ofhaving more than seven (7) initial biological entities is ##EQU5## whichis low, about 0.006. In summary for this illustration, the use ofmultiply occupied MDs with n=3 results in multiply occupied MDs forwhich only n=2 to 7 have significant probability.

Thus, for example, in contrast to relatively macroscopic preparationssuch as test tubes, cell culture flasks and microtiter wells which areoften used with thousands of biological entities such as cells, andwhich therefore usually result in small effect on the total populationby one biological entitiy, in the present example it is highly probablethat a single biological entity will have seven or fewer otherbiological entities within the same MD. For this reason, an unusualproperty of such a single biological entity, such as significantlygreater secretion rate, can have a large fractional effect, and therebybe measured, although non-optimally, in the presence of the otherbiological entities within the same MD.

It is also possible to straightforwardly carry out measurements onmultiple MDs, that is groups or clusters of MDs, wherein one or more ofthe MDs are multiply occupied. Such a process of measuring multiplemultiply occupied MDs is equivalent to measuring one large MD of sametotal volume as the sum of the MD volumes of the MDs which comprise thegroup or cluster of MDs, and can have the advantage of providing stilllarger throughput measurement rates while still retaining otheradvantages of MDS, such as the generally short characteristic diffusiontime, t_(D).

Manipulation of Microdroplets Surrounded by Non-Aqueous Fluids

It is generally desirable to provide means for applying forces on MDs,so as to provide means for manipulating MDs, such as changing ormaintaining the locations of MDs, so that external influence fromphysical, chemical and biological sources can be provided, for purposessuch as measurement, incubation and isolation. It is useful todistinguish two classes of processes for applying forces whichaccomplish changing or maintaining positions of MDs. These two classesare forces which depend on MDs being surrounded by a non-aqueous fluid,and forces which depend on MD force coupling entities, or intrinsicproperties of MDs, which are surrounded either by a non-aqueous fluid oran aqueous fluid.

In the case of MDs which are GMDs, following GMD creation in anon-aqueous fluid, the GMDs are left suspended, allowed to settle, orcaptured on a grid or filter, while surrounded by the non-aqueous fluid,such that the GMDs can now be manipulated through the use ofphysiochemical interactions that exploit differences in properties ofthe aqueous GMD interior fluid and the GMD-surrounding non-aqueousfluid.

Alternatively, if GMDs are created in an aqueous fluid, the GMDs can betransfered to a non-aqueous fluid, wherein the GMDs are manipulated bythe use of physiochemical interactions that exploit differences inproperties of the aqueous GMD interior fluid and the GMD-surroundingnon-aqueous fluid.

In this invention MDs can be surrounded by non-aqueous fluids in orderto provide a non-aqueous environment which surrounds or suspendsmicrodroplets Representative suitable non-aqueous fluids include liquidhydrocarbons, mineral oils, silicone fluids, ferrofluids, and heavyalcohols. This invention further involves physical manipulation ofmicrodroplets surrounded by a non-aqueous fluid so as to change theposition of such microdroplets by applying one or more physical forces,which are due to differences in properties of the MDs and thesurrounding non-aqueous fluid.

More particularly, such forces can be applied by selecting forcesselected from the group consisting of electrical force, magnetic force,field flow sedimentation fractionation force, acoustic force, opticalpressure force, gravitational force, sedimentation force, non-rotationalacceleration force, centrifugal force and centripetal force. While mostof these forces are extremely well known in general, the term opticalpressure force as applied to particles the size of MDs is more recent(see, for example, Ashkin et al, Nature 330: 769-771, 1987). In theprocess of this invention it is preferred to utilize electrical forceselected from the group consisting of electrophoresis force,iontophoresis force, electrokinetic force, dielectric force andcoulombic force, which are well known forces which can be applied toentities with electrical charge and/or dielectric properties whichdiffer from the dielectric properties of the medium surrounding theentities.

A particular process involving such electrical force is carried outusing the following steps: (a) providing a non-aqueous fluidenvironment, (b) providing a plurality of charged electrodes within saidnon-aqueous fluid environment wherein at least two electrodes are ofopposite polarity, (c) injecting an electically charged material capableof forming electrically charged microdroplets into the non-aqueousfluid; and (d) moving one or more charged microdroplets by means of anelectrical force associated with potential differences which are appliedbetween two or more electrodes, such that it is possible to producemicrodroplets within a non-aqueous fluid, to introduce microdropletsinto a non-aqueous fluid, to move microdroplets within a non-aqueousfluid, and to remove microdroplets from a non-aqueous fluid.Alternatively, MDs formed by any means and surrounded by a lowelectrical conductivity medium can be charged by contacting the MDs witha charged electrode.

It is thus possible to use such forces to manipulate the position of MDswith respect to measurement entities such as light emitting diodes,lasers, electrical capacitors, electrical inductors, electricalresistors, thermistors, thermocouples, fiber optics, photodiodes,phototransistors, photocells, piezoelectric sensors, specific ionelectrodes, oxygen electrodes, carbon dioxide electrodes, pH electrodesand electrodes allowing dielectric property measurement. Morespecifically, such physical force is utilized to move MDs into proximityto such measurement entities, to maintain MDs in proximity to suchmeasurement entities, and to remove MDs away from such measuremententities.

In addition to physically manipulating MDs with respect to one or moresuch measurement entities, in the process of this invention physicalforce is also utilized to move MDs which are located within measurementapparatus such as light measuring instruments, light microscopes,fluorescence microscopes, luminometers, fluorometers, photometers,time-resolved fluorometers, image analysis systems, colorimeters,spectrofluorimeters, particle counters, particle measuring systems,photoacoustic instruments, acoustic absorption instruments, acousticmicroscopes, dielectric spectrometers, electrical impedance measuringinstruments, calorimeters, thermal property measurement instruments andpiezoelectric mass loading instruments.

Similarly, the process of this invention can be used to cause MDs tocome into contact with other MDs, or for contacted MDs to be separated.The former is particularly useful to enhancing collisions andcoalescence of LMDs, while the latter is particularly useful forseparating weakly adhering GMDs.

Likewise, the process of applying one or more forces to MDs surroundedby a non-aqueous fluid can be used to physical force is used to providephysical manipulations of microdroplets selected from the groupconsisting of moving microdroplets into proximity of, maintainingmicrodroplets in the proximity of, and removing microdroplets away fromso that such MDs are exposed to external sources of physical influence,or exposed to sources of chemical influence. Such MDs surrounded by anon-aqueous fluid can be exposed to any physical source of externalinfluence, including sources of heat, electric fields, magnetic fields,electromagnetic radiation, optical radiation, ionizing radiation,acoustic radiation and acceleration. Suitable sources of chemicalinfluence include those which produce, release, modify or consumechemical compounds which are capable of dissolving in both thesurrounding non-aqueos fluid and the aqueous interior of such MDs.

Finally, all of such manipulations are of particular utility in the casethat at least one of the microdroplets contains at least one biologicalentity such as small multicellular organisms, groups of cells,individual cells, protoplasts, vesicles, spores, organelles, parasites,viruses, nucleic acid molecules, antibody molecules, antigen molecules,and aggregates of molecules.

In another embodiment of this invention it is possible to chemicallymanipulate MDs which are surrounded by a non-aqueous fluid. The presentembodiment relates to the chemical manipulation of MD interior fluidcomposition while the MDs are maintained surrounded by a non-aqueousfluid. This invention thereby allows biological entities withinnon-aqueous fluid surrounded MDs to be exposed to water soluble agentsand compounds, and allows water soluble chemical reagents to be added tosuch MDs, thereby significantly extending the ability to carry outbiological and chemical assays and tests using MDs.

More specifically, this invention consists of a process for deliveringwater soluble, entities and water insoluble entities, through anintervening non-aqueous fluid into MDs, with means for subeqentlydetermining the quantitiy of material so delivered. The major types ofdelivery processes involve:

(1) Introduction of LMDs or GMDs which contain (a) water soluble speciesto be delivered, and (b) water soluble optical indicator species whichallow subsequent measurement of the amount water soluble materialdelivered to each MD, or

(2) Dissolution of ampiphillic species (soluble in both aqueous andnon-aqueous fluids) into the surrounding non-aqueous fluid, such thatstirring and diffusion within the non-aqueous fluid provides essentiallyhomogeneous distribution of the ampiphillic species in the non-aqueousfluid, which in turn allows partitioning of such species into thenon-aquoeus surrounded MDs.

A more complete disclosure of these processes is provided in thefollowing sections. If not already surrounded by a non-aqueous fluid, itis first necessary to surround the microdroplets with an environmentwhich comprises a non-aqueous fluid

The general process of this embodiment involves chemically manipulatingMDs, which are surrounded by a non-aqueous fluid, by altering theconcentration of at least one chemical compound within the MDs byaltering the composition of the surrounding non-aqueous fluid. A generalmeans for accomplishing such chemical manipulation involves thedissolution of at least one chemical compound in the non-aqueous fluid,such that the chemical can partition into the aqueous interior of theMDs, and thereby cause a change in the chemical composition of theaqueous interior of the MDs.

Alternatively, the composition of the non-aqueous fluid can be alteredby adding MDs to the non-aqueous fluid, such that the MDs contain atleast one water soluble chemical compound. A general procedure foraccomplishing such chemical manipulation comprises the steps of: (a)dissolving at least one chemical compound in a first non-aqueous fluid,(b) contacting said first non-aqueous fluid with a second non-aqueousfluid, said second non-aqueous fluid surrounding at least onemicrodroplet, said chemical compound being soluble in the firstnon-aqueous fluid, in the second non-aqueous fluid, and in aqueousmedium, and (c) allowing time for partitioning of said chemical compoundfrom the first non-aqueous fluid into the second non-aqueous fluid, andsubsequently into at least one microdroplet.

Often it is desirable to use the additional step of mixing in order toshorten the time needed for partitioning of chemicals compounds intomicrodroplets from the non-aqueous phase, as such mixing reduces oreliminates the large concentration gradients that arise upon dissolvinga chemical compound in the non-aqueous fluid

Another general process for accomplishing chemical manipulation of MDssurrounded by a non-aqueous fluid involves providing additional MDs inthe non-aqueous fluid, such that the additional MDs contain thechemicals which are to be supplied to the original MDs. Such additionalMDs can be provided in the form of an emulsion, by contacting theemulsion to the non-aqueous fluid, such that the non-continuous phase ofthe emulsion comprises the additional MDs, and such that the continuousphase of the emulsion is comprised of the same, or a miscible,non-aqueous fluid as that which originally surrounds the original MDs.Often it is desirable to improve this process by mixing the emulsion andnon-aqueous fluid after the emulsion and non-aqueous fluid arecontacted.

Another version of this process involves the use of an emulsion whereinthe non-continuous phase of the emulsion consists of GMDs, rather thanLMDs.

Although in some applications it is not necessary to know how much of achemical compound is delivered to MDs, in some applications, for examplethe delivery of an antimicrobial, it is important to know the amount orconcentration of the chemical following the chemical manipulation of theMDs. In order to provide means for such measurement, it is useful toprovide at least one tracer compound with measurable properties which isdelivered to MDs by the same means as the chemical compounds, such thatMDs can then be measured for the amount of tracer compound In addition,a measurable tracer can also be provided in the non-continuous phase ofan emulsion contacted with the non-aqueous fluid, as this provides ameasure of the total amount of the emulsion so contacted with thenon-aqueous fluid Overall, the use of such tracers is important in thatmeasurement of the amount of at least one tracer compound allowsdetermination of the amount of at least one other chemical compoundwhich has been delivered to the MDs.

In order to measure such tracers, the tracer compounds are selected tohave measureable properties selected from the group consisting ofoptical properties, mass density properties, acoustic properties,magnetic properties, electrical properties and thermal properties. Ofthese, it is preferred to utilize tracers with optical properties suchas light scattering, light absorbance or colorimetric, fluorescence,time-delayed fluorescence, phosphorescence and chemiluminescence Aparticularly useful optical property is fluorescence, which can beprovided by using tracer compounds such as fluorescein, rhodamine,coumarin, lucifer yellow, phycoerythrins and their chemical derivatives

In order to enhance collisions and contact between the non-continuousphase of the emulsion and the MDs, it is possible to electrically chargethe non-continuous phase of the emulsion, for example, by using meanssimilar or identical to those for forming MDs with electrical charge byforcing an aqueous medium from an electrically conducting needle into anagitated non-aqueous fluid while a large electrical potential differenceis maintained between the needle at a large area electrode on theoutside of the non-aqueous fluid container

In conducting of the process of this invention, it is useful to alterthe chemical concentration within MDs by delivering chemical compoundshaving properties such as antiviral activity, enzyme inhibitoryactivity, antimicrobial activity, antifungal activity, cytotoxicactivity, and chemotherapeutic activity It is further useful to delivercompounds which are reactants for one or more enzyme catalyzedreactions, and it is particularly useful to utilize reactants such asfluorescein-di-β-D-galactopyranoside, resorufin-β-D-galactopyranoside,fluorescein diacetate, carboxyfluorescein diacetate, fluoresceinisothiocyanate diacetate, fluorescein digalactoside, 4-methylumbelliferone butyrate, 4-methyl umbelliferone phosphate, 1-naptholphosphate, 2-napthol phosphate, 3-μ-methyl-fluorescein phosphate,napthol AS phosphates, diacetyl 2-7-dichloro fluorescein, homovanillicacid, homovanillic acid+rhodamine lead, nicotinamide adeninedinucleotide (NAD) and resazurin

Microdroplet Incubation

Incubation consists of providing conditions for a time interval suchthat biochemical and biological reactions have an opportunity to occur.Incubation includes biochemical reactions and processes relating toreplication of genetic material, synthesis of biological material,degredation of biological material, metabolism, secretion, uptake,ligand binding, aggregation based on specific binding such as occurs inantibody-antigen reactions, the reactions which comprise the formationand/or growth of molecular complexes and aggrgates, and the complexreactions which comprise growth of virus, cells and small mutlicellularentities. Thus, during incubation biological entities have anopportunity to increase in size and/or number, and also to exhibitbiochemical activity. Further, by altering the concentration of one ormore compounds exhibiting properties such as antiviral activity, enzymeinhibitory activity, antimicrobial activity, antifungal activity,cytotoxic activity, and chemotherapeutic activity compounds, before,during or after an incubation, such compounds can be allowed to affectbiological entities.

The term biological entity refers to small biological structures whichare capable of being incorporated into liquid microdroplets and/or gelmicrodroplets, and includes small multicellular organisms, groups ofcells, individual cells, protoplasts, vesicles, spores, organelles,parasites, viruses, nucleic acid molecules, antibody molecules, antigenmolecules, and aggregates of molecules. Small multicellular organismsinclude fertilized eggs, blastomers, embryos, small nematodes and smallhelminths, which are of a size that can be incorporated into GMDs.Groups of cells include colony forming units, or CFUs, of naturallyaggregating cells, and also microcolonies that result from growth ofcells following one or more incubations. General types of cells ofinterest include animal cells, plant cells, insect cells, bacterialcells, yeast cells, fungi cells and mold cells. Organelles includemitochondria, choroplasts, ribosomes and lysosomes. Protoplasts includethose made from cells with cell walls by enzymatic digestion and otherwall removing processes, or by mutants which lack cell wall synthesisability. Virus includes those such as Herpes simplex, cytomegalo virus,Epstein-Barr virus, adenoviruses, influenza A or B virus, parinfluenza1,2 or 3 virus, mumps virus, measles virus, coronavirus, poliovirus,coxsackie A and B virus, echovirus, rhinovirus and hepatitis A and Bvirus, and human immunodeficiency virus or HIV. Nucleic acids includeboth DNA and RNA. Antibody molecules include IgA, IgG, and IgM obtainedfrom animals such as human, mouse, rat, goat, dog, pig and monkeys.Antigen molecules include large antigenic molecules with multipledistinct epitopes and/or overlapping epitopes, and small molecules suchas haptens. Aggregates of molecules include immunoprecipitates, antigenswhich have bound one or more antibodies, with or without labels,hybridized nucleic acids and non-covalently bound complexes of two ormore molecules. Although many of the examples of use of biologicalentities with microdroplets are described in terms of cells,particularly cells such as animal cells, plant cells, insect cells,bacterial cells, yeast cells, fungi cells and mold cells, the broadergroup is intended. Further, as used here, biological entity also refersto any of these previously mentioned entities which have been reactedwith one or more labling molecules, stains, dyes or with one or moreintervening molecules.

Capturing Molecules at Binding Sites in GMDs

This invention relates to GMDs which contain one or more providedbinding sites within the GMDs. Such GMDs are used to capture moleculeswithin the GMDs at the provided binding sites. Such GMDs, and processescarried out with such GMDs, allow important measurements, manipulationsand isolations to be carried out. The process of capturing moleculesconsists of: (a) incorporating specific sites and biological entitiesinto GMDs, (b) allowing molecules released from biological entities inGMDs to move by diffusion, convection or drift within the GMDs, suchthat some molecules encounter the binding sites and are bound at suchsites, thereby capturing molecules released from biological entities atbinding sites within GMDs.

Molecules captured within GMDs by this process can then be measured, inorder to determine properties or behavior of biological entitiescontained within the GMDs. Useful measurement means for measuring thecaptured molecules include optical, weighing, sedimentation, field flowsedimentation fractionation, acoustic, magnetic, electrical and thermalmeans It is preferred to use optical measurements such as measurementsbased on light scattering, light absorbance or colorimetric,fluorescence, time-delayed fluorescence, phosphorescence andchemiluminescence.

If the captured molecules have one or more measureable opticalproperties, the captured molecules can be measured by measuring anaturally occuring optical signal associated with the capturedmolecules, including optical signals such as light scattering, lightabsorbance or colorimetric, fluorescence, time-delayed fluorescence,phosphorescence and chemiluminescence.

In the practice of this invention it is often useful to provide one ormore incubations, in order to allow different conditions to effect theproduction and/or release of molecules from the biological entities. Forexample, it is useful to provide one or more incubations in order toallow molecules released by secretion from non-growing cells toaccumulate in sufficient numbers at binding sites that measurement ofthe captured molecules is more readily accomplished. Likewise, it isoften useful to provide one or more incubations which provide growthconditions for biological entities, particularly cells, in order thatcells can increase in size and/or number, such that the total ability ofthe biological entities within a GMD to produce and secrete moleculeswithin a GMD is increased, thereby resulting in capture of moremolecules at binding sites within such GMDs.

Gel microdroplets can also be physically isolated on the basis ofmeasurement of the captured molecules, followed by release, if desired,of biological entities from the isolated GMDs by processes such asdissolution of GMDs, mechanical disruption of GMDs and outgrowth by thebiological entities, such that biological entities contained within theisolated GMDs are physically isolated based on the measurement ofcaptured molecules. Examples of physical isolation include removing GMDsfrom a suspension and placing the GMDs in another suspension, sortingthe GMDs by using a flow cytometer/cell sorter, identifying GMDs bymicroscopy and utilizing micromanipulation to remove the GMDs, and usingoptical pressure to move GMDs to a known location. Following physicalisolation of GMDs, biological entities contained within the GMDs can bereleased from GMDs by processes such as dissolution of the gel matrix,mechanical disruption of the gel matrix and outgrowth from the gelmatrix by at least one biological entity

In some uses of this invention, a measurement of captured molecules isomitted, and instead forces which interact with one or more capturedmolecules are used to physically isolate GMDs, and thereby thebiological entities contained therein. This can be followed by releaseof biological entities from the isolated GMDs by processes such asdissolution of GMDs, mechanical disruption of GMDs and outgrowth by thebiological entities, such that biological entities contained within theisolated GMDs are physically isolated based on physical forcesinteracting with the captured molecules.

In cases wherein it is desired to measure captured molecules, thecaptured molecules do not necessarily have properties which allowsatisfactory measurement. In this case it is often possible to provide asubsequent step which comprises exposing GMDs to one or more labelingmolecules which have measurable properties and also are capable ofbinding to, and thereby labeling, the captured molecules. Following theprocess of exposing GMDs to one or more labeling molecules, the labelingmolecules are then measured, using either physical or chemical means.Examples of labeling molecules include antibodies, antigens, nucleicacids, lectins, receptors, enzyme inhibitors, and protein A, all withmeasurable labels.

In the practice of this invention it is preferred to use opticalmeasurements to measure labeling molecules which have bound to thecaptured molecules, including optical measurements of labeling moleculeswith labels which can be measured by light scattering, light absorbanceor colorimetric, fluorescence, time-delayed fluorescence,phosphorescence and chemiluminescence. Exemplary labeling moleculeswhich are labeled with such labels include antibodies, antigens, nucleicacids, lectins, receptors, enzyme inhibitors, and protein A, all withmeasurable labels.

Physical isolation of GMDs, and the biological entities contained withinthe GMDs, can be accomplished by using labeling molecules having a labelcapable of coupling with a physical force, such that following exposureof GMDs to labeling molecules, the GMDs with labeled captured moleculesare manipulated by at least one physical force in order to physicallyisolate such GMDs. In this embodiment it is preferred to provide and usemagnetic labels

It is often desirable to expose GMDs containing captured molecules toone or more intervening molecules before exposing, or simultaneouslywith exposing the GMDs to labeling molecules. This is accomplished by:(a) supplying at least one intervening molecule type and (b) supplyinglabeling molecules, such that intervening molecules bind to capturedmolecules and labeling molecules bind to intervening molecules. Examplesof intervening molecules include antibodies, antigens, nucleic acids,lectins, protein A and avidin. For use as an intervening molecule, it ispreferred to use unlabeled antibodies obtained from an animal speciesdifferent than that from which a labled antibody is obtained as alabeling molecule. For example, a mouse antibody to a captured moleculecan be used as an intervening molecule, and a fluorescence-labeled goatanti-mouse antibody can be used as a labeling molecule for theintervening molecule, such that a first exposure to this interveningmolecule, and a second or simultaneous exposure to the labeling moleculeresults in the ability to measure the captured molecules.

If desired, an intervening molecule can also have one or more labels,such that the binding of both intervening molecules and labelingmolecules increases the amount of label associated with capturedmolecules, thereby enhancing the measurement of captured molecules

It is useful to practice this invention using the biological entitiesincluding cells, spores, protoplasts, vesicles and small multicellularorganisms, and it is preferred to use small multicellular organisms andcells such as animal cells, plant cells, insect cells, bacterial cells,yeast cells, fungi cells and mold cells.

In cases in which biological entities do not secrete molecules ofinterest, or secrete molecules at a less than desired rate, it is usefulto cause biological entities to release cells by providing one or moreexternal stimuli. A general method for providing stimulus to cells,vesicles, protoplasts and small multicellular organisms involves theapplication of an electromagnetic stimulus which results inelectroporation (see, for example, Sowers and Lieber, FEBS Lett. 205:179-184, 1986)

Determination of Biological Growth

Prior use of GMDs has been based on determination of cell activity, moreparticularly metabolic activity, of one or more biological entitiescontained within the very small volume of a cell-occupied GMD. A generalfeature of such activity based determinations is that GMDs are providedwith a permeability barrier, in the form of a coating, or by suspendingthe GMDs in mineral oil (see Weaver et al., Ann N.Y. Acad. Sci., 434:363-372, 1984; Weaver, Biotech. and Bioengr. Symp. 17, 185-195, 1986;Williams et al., Ann. N.Y. Acad. Sci., 501: 350-353, 1987). Activitybased determinations using GMDs are based on the extracellularaccumulation of cell products within the very small volumes of GMDs, andthe use of chemical indicators or chemical assays in combination withchanges in the extracellular environment within a very small volume. Thedeterminations are fundamentally based on a time integration of theproduction rate for cell products which are released into theextracellular environment, and which are retained within the very smallvolume of a GMD. Further details concerning the production of GMDs maybe found in U.S. Pat. Nos. 4,399,219, 4,401,755 and 4,643,968, each ofWeaver, the teachings of which are incorporated herein by reference.

In such prior use of GMDs cell growth itself has not been determined,but instead the accumulated effect of cell activity, which is due toboth to the activity per cell and to cell number, was the basis of thedeterminations. Thus, for example, an increase in cell products within aGMD with a permeability barrier could be due either the presence of onehighly active cell, such as a single yeast cell, or could be due to aninitial bacterium which rapidly grows to form a microcolony of severalcells, which microcolony has activity several times that of anindividual bacterium. In the first example, a single yeast cell, withoutthe occurrence of growth, is the basis of a determination, while in thesecond example, the determination occurs primarily because of theincrease in cell biological material, and corresponding increasedactivity, due to growth. As demonstrated by this illustration, fordifferent cell types the same change in extracellular chemicalconcentration due to activity can occur with or without growth,depending on the relative individual cell activity and the relative cellgrowth rate, so that for this reason the prior use of GMDs does notprovide a general means for determining cell growth. Further, although asignificant advantage of the prior use of GMDs is that cell growth isnot necessarily the basis of determinations, generally the type ofactivity must be known. For example, although a great manymicroorganisms produce significant amounts of acids, allowing GMDdeterminations based on extracellular pH changes within the very smallvolume of a GMD with a permeability barrier, the production ofsignificant acid is not a universal property of cells. For this reason,prior use of GMDs has required knowledge of the type of biochemicalactivity exhibited by a cell, whereas, in contrast, the presentinvention is much more general.

For the above reasons, neither the previous methods of others, nor theprevious use of GMDs to detect cells based on metabolic productretention, actually provides a rapid determination of cell growth, afundamental process in which biological entities increase in biologicalmaterial and/or in number. In contrast, the present invention provides ageneral means for biological entity growth determination, whereinentrapment of initial and progeny biological entities within MDs,preferably GMDs surrounded by an aqueus fluid or medium, is combinedwith measurement of the biological material within MDs, and which,because of the very small size MDs, allows incubation conditions andanalysis conditions to be changed rapidly because of the small diffusiontimes within MDs. In the case of MDs which are GMDs, this invention alsoallows optical measurements to be made with small optical path lengthswithin a gel matrix. This reduces optical measurement error associatedwith light scattering, light absorbtion or autofluorescence in a gelmatrix.

In the case of cells which naturally aggregrate and adhere to each otherin small numbers, the present invention determines the growth of colonyforming units (CFUs).

In the general practice of this invention MDs are formed by any of thepreviously described means, such that biological entities such as smallmulticellular organisms, groups of cells, individual cells, protoplasts,vesicles, spores, organelles, parasites, viruses, nucleic acidmolecules, antibody molecules, antigen molecules, and aggregates ofmolecules are incorporated into MDs. The resulting MDs can containbiological entities such that MDs of different sizes have a highprobability of being mostly unoccupied, individually occupied ormultiply occupied. Measurement of biological material is thenaccomplished by making measurements on the MDs, either individually orin small groups, such that measurements responsive to biologicalmaterial are made. Biological material consists of the constituativemolecules and structures found in biological entities such as smallmulticellular organisms, groups of cells, individual cells, protoplasts,vesicles, spores, organelles, parasites, viruses, nucleic acidmolecules, antibody molecules, antigen molecules, and aggregates ofmolecules. Thus, examples of biological material include proteins,nucleic acids, phospholipids, polysaccharides, enzymes, antibodies, cellreceptors and other well-known biological molecules.

Measurement of biological material can be accomplished by usingmeasurements based on optical, weighing, sedimentation, field flowsedimentation fractionation, acoustic, magnetic, electrical and thermalmeans. Such measurements can be based on properties such as opticalproperties, mass density properties, acoustic properties, magneticproperties, electrical properties and thermal properties. It ispreferred to utilize measurements based on optical properties such asare measureable utilizing light scattering, light absorbance orcolorimetric, fluorescence, time-delayed fluorescence, phosphorescenceand chemiluminescence. Thus, useful measurement apparatus includes flowcytometry apparatus, flow-through-microfluorimetry apparatus, opticalparticle analyzers apparatus, fluorescence microscopy apparatus, lightmicroscopy apparatus, image analysis apparatus and video recordingapparatus.

In some cases naturally occurring optical properties of the biologicalmaterial of biological entities can be used to measure the biologicalmaterial, as this provides a naturally occurring optical signal Examplesof such naturally occurring signals are light scattering, lightabsorbance or colorimetric, fluorescence, time-delayed fluorescence,phosphorescence and chemiluminescence. However, in order to enhance themeasurement of biological material in GMDs it is often useful to employstaining protocols which utilize stains such as stain indicative ofbiological composition, stain indicative of enzyme activity, and stainindicative of cell membrane integrity, as such stains are responsive tothe amount, activity and/or state of biological material. Morespecifically, it is often desirable to use stains in the generalcatagories of fluorescent stains, light absorbance stains and lightscattering stains, and more specifically stains such as nucleic acidsstains, protein stains, lipid stains, cell membrane stains, cell wallstains, stains responsive to enzyme activity, stains responsive totransmembrane potentials and cell surface receptor stains. Still otheruseful types of stains include transmembrane potential stains, membraneexclusion stains and intracellular enzyme activity responsive stains.

It is generally preferred to use fluorescent stains such as propidiumiodide, ethidium bromide, FITC (fluorescein isothiocyante), fluoresceindiacetate, carboxyfluorescein diacetate and FITC diacetate. Many othersuitable fluorescent stains for biological material are well known (see,for example, Haugland Handbook of Fluorescent Probes and ResearchChemicals, Molecular Probes, Junction City).

In the general practice of this invention it is preferred to makemeasurements of MDs involving predominantly measurements of individualor single MDs However, measurements can also be made simultaneously ongroups of two or more MDs. Likewise, it is preferred to makemeasurements on MDs consisting predominantly of microdroplets with ahigh probability of containing less than two biological entities priorto incubation, but measurements can also be made on microdropletsspecimens consisting predominantly of microdroplets with a highprobability of containing at least two biological entities prior toincubation.

Although it is useful to measure the amount of biological material inGMDs without requiring measurement of the volumes of individual GMDs, orof groups of GMDs, it is often useful to measure such GMD volumes. Thisdata can be used to provide the basis for further analysis orinterpretation of the biological material measurements. For example,measurement of such GMD volumes provides the basis for determining thevolume of a sample which was contained in the measured GMDs, as themeasured GMD volumes can be summed to yield the total volume analyzed,with correction for dilution if needed.

Additionally, it is often useful to analyze the measurement of MDvolumes, or volumes of groups of MDs, with statistical formulae whichrelate the occupation of MDs to MD volume and the average concentrationin the suspension or solution from which MDs were formed. Morespecifically, it is often useful to analyze MDs and measurements of theamount of biological material, such that measurement of the biologicalmaterial provides a basis for classifying each MD, or group of MDs, asbeing unoccupied or occupied. It is then useful to further analyze theMDs and measurements by using Poisson statistics formulae and modifiedPoisson statistics formulae.

If control analysis is desired, one or more specimens of MDs aremeasured without incubating. Other MDs are then exposed to conditionsfor which growth determination is sought, and incubated for one or aplurality of incubation periods, and some or all of the MDs thenmeasured, individually or in groups, for the amount of biologicalmaterial present in the individual MDs or in the groups of MDs. Theresults of such MD measurements are interpreted as the amount of changein biological material during the control or incubation periods, andsuch change in biological material is attributed to the amount of growthwhich occured during the incubation period.

Following such measurement of the change in the amount of biologicalmaterial, one or more forces can be applied such that MDs containingbiological material with a first change in the amount of biologicalmaterial are isolated from MDs not containing biological entities withthe first change in the amount of biological material

In the preferred embodiment of the invention, MDs are created with awide range of sizes, such that some range of sizes has a highprobability of containing zero or one initial biological entities. Onesubpopulation of MDs is measured without incubation in order to providea control, and one or more other subpopulations of MDs are incubatedunder desired conditions and then measured individually for the relativeamount of biological material in each MD.

It is useful to separately consider the case wherein the volume, V_(MD),of each MD is known or measured, and it is also known that the samplecontains biological entities at approximately known concentration,ρ_(S), so that following concentration or dilution of the sample, andafter addition of gelable material, the resulting concentration, ρ, isknown, and is the suspension from which MDs are made.

A preferred embodiment of this invention involves formation of MDs whichare GMDs, followed by suspending these GMDs in an aqueous or non-aqueousmedium. Following one or more incubations of these GMDs the growth ofindividual biological entities into microcolonies of two or morebiological entities can occur. In the case in which GMDs are surroundedby a non-aqueous fluid, the GMDs are transfered from the non-aqueousfluid into an aqueous fluid. A staining procedure for biological entitybiological material is then used to provide the basis of one or moreoptical measurements, such that the magnitudes of the optical signalsprovides a determination of the biological material present in eachmeasured GMD.

In a representative case of the general invention, wherein MDs are GMDssuspended in an aqueous medium, the biological entities are cells suchas animal cells, plant cells, insect cells, bacterial cells, yeastcells, fungi cells and mold cells. A fluorescent dye is used to stainnucleic acids, the fluorescence associated with the dye is measured inthe GMDs, preferably in individual GMDs, and thefrequencey-of-occurrence of a certain magnitude of a fluorescence signalcan be displayed as a function of the magnitude of fluorescence signal.This type of plot is generally termed a histogram. Such analysis showsthat incubated GMDs with growing cells produce histograms withfluorescence peaks that occur at larger magnitude fluorescence. It isoften convenient to plot the frequency-of-occurrence versus logarithmfluorescence, because cells growing in the exponential phase thenexhibit a linear or proportional increase in the average logfluorescence as a function of time. It is found that such plotsgenerally exhibit peaks with increasingly larger average fluorescencefor GMDs incubated for longer times. The area of such peaks provides ameasure of the average growth of cells, and thereby the average growthrate. It has been found that such GMD based average growth ratedeterminations compare favorably with conventional, total populationmethods. The present invention also provides a measure of the variationin growth rate, or growth rate distribution, which is reflected in thevariation of fluorescence within the fluorescence peaks which is greaterthan the variation or instrumental error in the fluorescence measurementapparatus itself, and which is not determined by conventional methods

This invention can also be used to determine the lag time in growth ofbiological entities. This lag time is the delay in achieving anexponential growth rate following exposure to changed conditions. Suchdeterminations are made by comparing the magnitude of the change in theamount of biological material in GMDs incubated under particular changedconditions to the change in the amount of biological material in otherGMDs incubated under control conditions. Such lag time determinationsutilize the growth rate determinations described elsewhere in thisdisclosure.

Further, in some cases such analysis reveals a fluorescence peak atapproximately the location of the initial or non-incubated peak, whosepeak area provides a measure of non-growing cells for the incubationconditions used. If such conditions correspond to conditions whichordinarily support growth of the cell type, then the ratio of the numberof GMDs with growing cells to the number of both growing and non-growingcells can be interpreted as the cloning or plating efficiency in GMDsfor these conditions. Conventional rapid growth measurements based onthe combined effects of many cells do not provide direct determinationof non-growing cells, but instead can only provide a determination basedon the combined effects of growing and non-growing cells, and therelative numbers of growing and non-growing cells is not known a priori.

In addition to measuring the biological material consisting of the totalamount of one or more types of biological material, it is often usefulto supply chemical compounds in order to utilize reactions such asdegrading reactions of stainable biological material and blockingreactions of stainable biological material, so that enhanced measurementis achieved. For example, in the case of GMDs in an aqueous mediumwherein it is useful to measure total stainable nucleic acid to providea measurement of growth, it is also possible, for biological entitiesthat contain DNA which is replicated, to provide a RNA degredationprocess such as exposing the GMDs to RNAase, which degrades the RNAwhile leaving the DNA, such that subsquent exposure to a necleic acidstain allows DNA to be measured in GMDs. Such measurement of DNA thenprovides the basis for determining replication of biological entities.

The process of the present invention can provide determinations ofbiological entity growth which are based on measurement of biologicalmaterial within MDs which initially had a high probability of havingzero or one biological entity. This biological entity growthdetermination process is rapid, often requiring about one averagegeneration or doubling time. However, the process can also beadvantageously carried out using more generations or doublings, in orderto allow possibly unstable cells to cease growing, and/or in order toutilize less sensitive and less expensive optical measurement apparatus.

Although the previous illustration describes a preferred embodiment, inwhich there is a high probability of some MDs, of some size or volumerange, V_(MD), being unoccupied or individually occupied it is alsopossible to make growth measurements using MDs in a size range, relativeto the biological entity suspension concentration, ρ, for which there isa moderate or high probability of multiple occupation. In this case themeasurement of optical signals from individual MDs relates to the totalbiological material within each MD, and therefore correponds to anaverage growth determination, wherein the average is over the smallnumber of biological entities initially present for each MD size. Forexample, if the biological entity concentration, ρ, just prior to MDcreation results in n=3 biological entities in MDs of size V_(MD), thenthe average growth determination has a high probability of correspondingto the average growth behavior of 3 biological entities, and can revealsome aspects of growth heterogeneity or variability which cannot bedetermined by conventional methods which are based on the combinedeffects of many, typically 10⁴ or more, biological entities.

Alternatively, in a related embodiment, MDs with a high probability ofinital occupation by zero or one biological entities can be measured insmall groups rather than individually. This can be advantageous, forexample, in flow cytometer measurement of GMDs suspended in an anqueousmedium, wherein measurement conditions allow several GMDs to be presenttransiently in the optically measured region, and thereby allows ahigher rate of GMD analysis. Likewise, this can be advantageous inmeasurement using microscopy, of MDs suspended in, or surrounded by, anonaqueous fluid, wherein measurement conditions allow several MDs to bepresent in the measurement field of view, and thereby allows a higherrate of MD analysis. In this embodiment, measurements on groups of MDsincubated for two or more incubation periods are compared, and thedifferences in optical signals from the groups of MDs are interpreted asresulting from differences in biological entity material within thegroups of MDs. These differences are interpreted as arising frombiological entity growth within the groups of MDs. For example,measurements on groups of incubated MDs are compared to measurements ongroups of non-incubated MDs, such that differences in optical signalsfrom the incubated groups of MDs and non-incubated groups of MDs areinterpreted as resulting from differences in biological entity material.These differences are further interpreted as resulting from biologicalentity growth during the incubation period. This embodiment is relatedto the previous embodiment, in that the biological material opticalsignal corresponding to several initial biological entities forms thebasis of the measurement, and therefore provides the average growthdetermination as having a high probability of corresponding to theaverage growth behavior of several biological entities. This method canthus reveal some aspects of growth heterogeneity or variability whichcannot be determined by conventional methods which are based on thecombined effects of many, typically 10⁴ or more, biological entities.

In the present invention, after creation, the MDs are placed in desiredconditions for determining growth. Typically MDs can be suspended inaqueous or non-aqueous media which provide a wide range of differentchemical compositions, and at different pH, temperature, partialpressures of oxygen and carbon dioxide, etc. This allows biologicalgrowth under a wide range of conditions to be determined. As analternative to suspension, MDs can be held stationary by allowing MDs tosediment under the influence of an applied force such as gravitationalor centripetal force. In the case of GMDs, the GMDs can be temporarilytrapped against a porous mesh or filter by a perfusing flow, whichprovides a supply of growth medium past the trapped GMDs.

The transport of chemicals within MDs is generally governed bydiffusion. Thus, in the case of MDs surrounded by a non-aqueous fluid,the supply and removal of chemicals to biological entities within theMDs is governed by the partioning of chemicals between the non-aqueousfluid and the interior aqueous fluid of the MDs, and is further governedby diffusion within the MDs. Because of the relatively small size ofMDs, the characteristic diffusion time, τ_(diffusion) =x² /D can beshort, as x is the characteristic distance over which diffusivetransport occurs, and γ_(MD) ≈x. This yields τ_(diffusion) from about 4min to about 5×10⁻⁶ sec for MDs with diameters of 1000μ to 0.2μ, andfrom about 1 min to about 6×10⁻³ sec for MDs with diameters of 500μ to5μ for small molecules with D≈10⁻⁵ cm² sec⁻¹. This value isapproximately representative of the size molecule which can readilypartition and diffuse between the non-aqueous fluid surrounding a MD andthe interior aqueous fluid of the MD. As a result, if the kinetics ofpartioning and transport within the non-aqueous fluid are not limiting,the characteristic diffusion time, τ_(D), governs changes, and can beshort. Thus, the concentration of some types of molecules, specificallythose with significant solubility in both the non-aqueous fluid and theaqueous interior medium can be changed rapidly in such MDs.

Similarly, the supply and removal of chemicals to biological entitieswithin GMDs surrounded by an aqueous medium is governed primarily bydiffusion, and by partitioning between the external aqueous mediumsurrounding the GMDs and the aqueous medium within the GMDs, because thegel matrix effectively clamps viscous flow, that is, increasesresistance to viscous flow. The partioning between the external aqueousmedium and the GMD interior medium is often non-selective, because manygels used to form GMDs do not exclude, absolutely or partially, mostchemicals of interest. In some cases, however, gel materials can havecharge or size exclusion properties so as to partically or absolutlyexclude some molecules from the interiors of GMDs.

Further, because of the relatively small size of GMDs compared toconventional, more macroscopic gel preparations, the characteristicdiffusion time, τ_(diffusion) =x² /D, where x is a characteristicdimension such as the thickness of a macroscopic gel slab and D is thediffusion constant within the aqueous liquid within the gel, can rangefrom shorter to much shorter than for conventional gel preparations.Depending on the type of biological entity used, and the chemicals ofinterest, τ_(diffusion) can have a wide range of values, as can be seenby using γ_(GMD) ≈x, which gives τ_(diffusion) from about 4 min to about5×10⁻⁶ sec for GMDs with diameters of 1000μ to 0.2μ, and from about 1min to about 6×10⁻³ sec for GMDs with diameters of 500μ to 5μ for smallmolecules with D≈10⁻⁵ cm² sec⁻¹, and about a factor of 100 longer formacromolecules with D≈ 10⁻⁷ cm² sec⁻¹. As a result, even theconcentration of macromolecules can be changed rapidly in GMDs withdiameters of about 200μ or less, as the corresponding diffusion time isabout 1 hour, a value much smaller than the doubling time of typicalmammalian cells. This value can be changed even more rapidly in thesmaller GMDs which can be used with smaller microorganisms such asbacteria and yeast, which microorganisms have shorter doubling times. Asa further example, 20μ GMDs used with rapidly growing bacteria for whichthe doubling time, t₂, is typically about 20 min, have an appropriatelyshort τ_(diffusion) of about 0.1 sec for small molecules and about 10sec for macromolecules.

After one or more incubation periods the MDs are measured, preferably byoptical means. Other measurement means include methods sensitive to massdensity, such as weighing, sedimentation, and sedimentation field flowfractionation, and additional methods based on acoustic, magnetic,electrical and thermal properties of MDs containing different amounts ofbiological material. Sedimentation field flow fractionation force can beprovided by simultaneously utilizing a hydrodynamic force and asedimentation force (see, for example, Levy and Fox, Biotech. Lab.6:14-21, 1988). Acoustic measurements utilize sound absorbtion andreflection of biological material, as is utilized in acoustic microscopy(see, for example Quate, Physics Today, August 1985, pp. 34-42).Magnetic measurement utilizes diamagnetic, paramagnetic and,occasionally, ferromagnetic properties of biological material. Thermalmeasurement utilizes thermal conductivity, thermal diffusivity andspecific heat properties of biological material (see, for example,Bowman et al, Ann. Rev. Biophys. Bioengr. 4: 43-80, 1975). Electricalmeasurement utilizes electrical resistance and dielectric properties ofbiological material, such that measurement of the dielectric propertiesat various frequencies can provide measurement of biological material(see, for example, Kell in Biosensors: Fundamentals and Applications,Turner et al (Eds), Oxford University Press, Oxford, pp. 427-468; Harriset al, Enzyme Microb. Technol. 9: 181-186, 1987). The measurement ofelectrical resistance of biological entities such as cells is well knownto provide a means for measuring cell size, and is the basis forparticle analyzers such as the Coulter Counter (see, for example, Kachelin Flow Cytometry and Sorting, Melamed et al (Eds), Wiley, New York, pp.61-104). In the present invention, the preferred electrical measurementis utilized with GMDs suspended in or surrounded by an aqueous medium,is preferably used with biological entities with bilayer membranes.These entities include small multicellular organisms, cells, vesiclesand protoplasts. The electrical measurement is based first on a rapiddiffusional exchange of medium within a GMD from a defined electricalresistance medium such as physiological saline, followed by a secondstep of passing GMDs though a particle analyzer such as a CoulterCounter. The gel matrix of such a GMD provides negligible electricalresistance compared to such biological entities, thereby allowingmeasurement of the amount of biological material associated with cellscontained in GMDs.

It is presently preferred to utilize optical measurements in order tomeasure biological material contained within MDs. Well known generaloptical measurements sensitive to biological material include lightscattering, light absorbance or colorimetric, fluorescence, time-delayedfluorescence, phosphorescence and chemiluminescence.

Biological material contained within MDs can in some cases be adequatelymeasured utilizing naturally occuring optical properties of thebiological material. Thus, for example, fluorescence of the biologicalmaterial, light absorbance by the biological material, and lightscattering by the biological material can sometimes be used.

Prior to optical measurements, however, it is often preferred to exposeMDs, particularly GMDs surrounded by an aqueous medium, to one or morestaining processes. These processes can be general (e.g. nucleic acidstains) or they can be specialized (e.g. fluoresence-labeledantibodies), depending on the type of biological entity and the purposeof the biological entity growth determination. In the case of MDssurrounded by a non-aqueous fluid, stains can be introduced through thesurrounding non-aqueous fluid by dissolving the stains in thenon-aqueous fluid, or by supplying the stains in the non-continuousphase of an emulsion which is contacted with the non-aqueous fluid. Inthe case of GMDs surrounded by an aqueous fluid or medium, stains can beintroduced through the surrounding aqueous fluid by dissolving thestains in the aqueous fluid.

In the general case wherein growth analysis without further use of thebiological entities is desired, any biological entity staining process,including those which kill biological entities, can be used. In the casewherein further analysis or use of viable biological entities isdesired, biological entity staining which allows biological entitysurvival is used. As is well known in the art, there are biologicalmaterial stains for nucleic acids stains, protein stains, lipid stains,cell membrane stains, cell wall stains, stains responsive to enzymeactivity, stains responsive to transmembrane potentials and cell surfacereceptor stains.

Following exposure of the MDs to suitable stains, the MDs areindividually measured, or the MDs are measured in small groups, providedthat the probability of finding more than one biologicalentity-containing MD in the group is low. In the exemplary case ofbiological entities stained by fluorescent compounds, optical analysissuch as digital fluorescence microscopy or flow cytometry is used tomeasure individual MDs, using a wavelength band sufficiently differentfrom that used for any detection of measurement of MD properties. Thismethod allows simultaneous, or serial, measurement of MD properties andof biological entities with said MD. The associated fluorescence signalsare acquired and measured, with correction for spectral overlap ifnecessary, by conventional means.

The relatively small size of MDs results in the possibility of moreflexible analysis. For example, conventional flow cytometers have flowbiological entity channel diameters of several hundred microns, whichprohibits the use of flow cytometry with conventional macroscopic gelpreparations, but which readily allows the use of MDs in the size rangefrom somewhat less than the flow biological entity channel size andsmaller.

The magnitude of the optical signal due to the biological entity stainin each MD, or group of measured MDs, is compared to the optical signalof individual biological entities, whether or not such individualbiological entities are entrapped in MDs, thereby providing acalibration. Comparison of the MD optical signal magnitude to that ofindividual biological entities provides the basis for determination ofgrowth of individual biological entities, for which the growthdetermination can often be made within one generation time, but withouta need for significant prior culture to obtain large numbers ofbiological entities.

By making a large number of such individual biological entity growthdeterminations, the distribution of growth rate, distribution of lagtime, and the plating efficiency can be automatically determined bycomputer calculation. Manual or visual inspection and scoring of MDs canalso be used, but is relatively labor intensive and therefore more proneto error, so that the preferred processes are those conducted usingautomated measurement means.

Determination of Effects of Compounds on Biological Entities

The present invention further provides means for determining importantproperties of chemical compounds and agents as said properties relate tothe effects of said compounds on biological entities, particularly thegrowth of biological entities. Alternatively, this invention alsoprovides means for determining important properties of biologicalentities, particularly cells, relating to the susceptibility orresistance of the biological entities to the effects of compounds oragents on behavior of the biological entities. This is especially usefulfor determining the effects of compounds on the growth behavior ofbiological entities which can be determined by measuring the amount ofbiological material associated with biological entities contained inMDs. A general process for determining the effect of compounds on thegrowth of biological entities comprises the steps of: (a) exposing MDsto at least one compound, said compound being such that its effect onthe growth of said biological entities is to be determined, and (b)measuring biological material within at least one MD In someapplications of this invention, MDs can be supplied which alreadycontain biological entities, but in other applications in is necessaryto first incorporate biological entities into gel microdroplets. Theincorporation can be accomplished using any of previously describedprocesses for the formation of MDs.

As used in this invention, the term specimen of microdropletsencompasses both specimens of LMDs and specimens of GMDs, and refers toa subset of the MDs formed from a sample. Thus, for example, if a sampleis processed so as to lead to the formation of about 10⁵ MDs, these MDscan be divided into ten approximately equal specimens of MDs whereineach specimen of MDs contains about 10⁴ MDs.

The effect of compounds or agents on biological entities is generallynot revealed instantaneously, but instead after a period of time hasbeen allowed to elapse, such that at least one incubation is generallydesirable in order to bring out the effect of of compounds or agents onbiological entities. Further, the effect of compounds or agents onbiological entities can often be advantageously determined by exposingat least two specimens of microdroplets to different concentrations ofthe compounds or agents. In order to interpret changes caused by theexposure of biological entities in MDs to compounds and agents, it isoften desirable that at least one specimen of MDs not be exposed to thecompounds or agents, thereby providing at least one control condition.

The effects of many compounds and agents on biological entities can bedetermined by this invention, including the effects of compounds such asantibiotics, antimicrobial compounds, antifungal compounds,chemotherapeutic compounds, toxic compounds, cytotoxins, irreversibleinhibitors, reversible inhibitors, mutagenic compounds, hazardouscompounds, hormones, growth factors, growth enhancers, nutrients,vitamins, food preservatives, pesticides and insecticides. Further, manydifferent types of biological entities can be used in this invention,including small multicellular organisms, groups of cells, individualcells, protoplasts, vesicles, spores, organelles, parasites, viruses,nucleic acid molecules, antibody molecules, antigen molecules, andaggregates of molecules. It is preferred to use cells such as animalcells, plant cells, insect cells, bacterial cells, yeast cells, fungicells and mold cells.

Additional flexibility can often be obtained by: (a) using at least twoincubation periods, and (b) using at least one change in theconcentration of the compounds between incubation periods. For example,if it is desired to test the reversability of a growth altering compoundor agent, a first incubation can be used with the compound or agentpresent, followed by a second incubation with the compound or agentabsent.

Control conditions can be provided by: (a) exposing a single specimen ofmicrodroplets to at least one change in concentration of at least onecompound or agent, (b) using a first incubation as a control for growth,and (c) using at least one subsequent incubation with exposure to thecompound or agent, thereby providing a serial process in which a controlis followed by exposure to compounds or agents Alternatively, a processcan be carried out by: (a) exposing at least one specimen ofmicrodroplets to at least one change in concentration of at least onecompound, (b) using at least one other specimen of microdroplets withoutexposure to said compound, in order to provide a control for growth, and(c) using separate incubation of at least one exposed specimen and onecontrol specimen.

More specifically, the process of this invention can be used todetermine the antimicrobial susceptibility of microorganisms to variouscompounds. Specimens of GMDs which contain one or more microorganismsare exposed to an antimicrobial at one or more concentrations, and thegrowth determined by using an incubation for each concentration. Bycomparing the amount of growth at different concentrations, theeffectiveness of the compound to inhibit growth of the microorganism atthese concentrations can be determined. This determination correspondsto a determination of the antimicrobial susceptibility of thatmicroorganism for the tested compound.

A related process provides determination of the sensitivity of cancercells to compouds such as chemotherapeutic compounds, in which case itis preferred to use GMDs. Specimens of GMDs which contain one or morecancer cells are exposed to a compound at one or more concentrations,and the growth determined by using an incubation for each concentration.By comparing the amount of growth at different concentrations, theeffectiveness of the compound to inhibit growth of the cancer cells atthese concentrations can be determined. This determination correspondsto a determination of the chemotherapeutic susceptibility of the cancercells to the tested compound. An advantage of the present invention isthat it is possible to make measurements on GMDs which have a highprobability of being occupied by a small numbers of cells, preferablyless than two cells. Further, measurements can be made on large numbersof GMDs which contain cells. As a result, for example, this inventionprovides the ability to determine the chemotherapeutic susceptibility oflarge numbers of individual cancer cells, for example 1,000 cells to10,000 cells. Thus, it is possible to determine the distribution ofamount of growth by a large number of cells, which growth is notnecesarily identical, thereby allowing the distribution in cancer cellgrowth to be determined. This, in turn, provides a determination of thedistribution of susceptibility of the cancer cells to the compound.Thus, for example, if a subpopulation comprising 10% of the cancer cellsis resistant to a compound, or mixture of compounds, significant growthwill be found in about 10% of the occupied GMDs. Such determinations canhave good statistical significance because a large number of individualGMD measurements can be made, wherein there is a high probability thatGMDs are occupied by less than two cells.

Continuing this example, if a resistant subpopulation comprising about10% of the cells is found on the basis of measuring 10⁴ cells, thenumber of GMDs occupied by resistant cells is about 10³. Thecorresponding statistical error in sampling, and therefore indetermining the size of the resistant subpopulation, is due to the errorin counting randomly occurring events. This statistical error is wellknown to be described by √N_(random) event, so that in this illustrationthe error is is √N_(resistant) cell =√10³ =32, which corresponds toabout 3% error, and is therefore highly accurate in determining the sizeof the resistant subpopulation.

Measurements of the amount of biological material in GMDs can providethe basis for measurement of growth, and other biological activity andfunction, by biological entities such as cells within GMDs. Suitablemeasurement means for measuring the biological material include optical,weighing, sedimentation, field flow sedimentation fractionation,acoustic, magnetic, electrical and thermal means. It is preferred to useoptical measurement means, particularly measurements based on lightscattering, light absorbance or colorimetric, fluorescence, time-delayedfluorescence, phosphorescence and chemiluminescence.

As described elsewhere in this disclosure, groups of microdropletsconsisting predominantly of single microdroplets can be simultaneouslymeasured, or, alternatively, groups of microdroplets consistingpredominantly of at least two microdroplets can be simultaneouslymeasured. As also described elsewhere, the measured microdroplets canconsist predominantly of gel microdroplets with a high probability ofcontaining less than two biological entities prior to incubationAlternatively, the measured microdroplets can consist predominantly ofmicrodroplets with a high probability of containing at least twobiological entities prior to incubation.

Finally, this invention can be used with a step wherein measurement ofgrowth is used to determine the effect of at least one compound ongrowth characteristic behavior selected from the group consisting ofplating efficiency, growth rate distribution, average growth rate,growth lag time distribution and average growth lag time

Enumeration of Viable Biological Entities

This invention involves also the determination of the number of viablebiological entities per volume, which comprises enumeration of viablebiological entities, a measurement which is widely used in biology andmedicine. For example, it is common to determine the number of viablemicroorganisms per ml of a fluid sample. In this process it is importantto make determinations which are rapid, based on the number ofmicroorganisms, and based on a stringent criterion for microorganismviability. As described herein, it is often possible to extend theconcept of growth to include the growth of biological entities such assmall multicellular organisms, groups of cells, individual cells,protoplasts, vesicles, spores, organelles, parasites, viruses, nucleicacid molecules, anti-body molecules, antigen molecules, and aggregatesof molecules. It is preferred, however, to carry out the process of thisinvention with cells such as animal cells, plant cells, insect cells,bacterial cells, yeast cells, fungi cells and mold cells, particularlyfor normal human cells, human cancer cells, pathogenic bacteria,pathogenic yeast, mycoplasms, parasites, and pathogenic viruses. Thisinvention provides a general means for rapidly enumerating suchbiological entities using the criterior viability based on growth, andalso, for some biological entities, using criteria provided by vitalstains.

More specifically, this invention provides means for determining thenumber of viable biological entities per volume of a sample, the processcomprising the steps of: (a) exposing at least one MD, which contains atleast one biological entity, to conditions for which the number ofviable biological entities is to be determined, (b) measuring biologicalmaterial in GMDs, (c) measuring the volumes of the associated GMDs, and(d) thereby determining the number of viable biological entities pervolume in the sample. In cases wherein MD volumes are not known, theadditional step of measuring MD volumes is used. Prior to making thisdetermination, it is necessary to incorporate biological entities intoMDs. This can be accomplished using previously described means forforming MDs with sample material which contains biological entities.

Although an indication of viability can, in some cases, particularly forcertain types of cells, be determined by use of vital stains such asmembrane potential responsive dyes, membrane exclusion dyes such as thelight absorbance dye trypan blue, and such as the fluorescent dyespropidium iodide and ethidium bromide, and intracellular enzyme/membraneintegrity dyes such as fluorescein diacetate, carboxyfluoresceindiacetate and fluorescein isothiocyanate diaceate, (see, for example,Shapiro, Practical Flow Cytometry, A. R. Liss, New York, 1985) it isgenerally desirable to use a more stringent criterion for determiningthat a biological entity is viable. Many biological entities,particularly cells and viruses, are stringently determined to be viableonly by determining that the biological entities are capable of growth,that is, of increasing in size and/or number. Thus, the presentinvention can be utilized to enumerate biological entities by a processwherein at least one incubation is provided, in order to provide anopportunity for growth prior to measurement of the amount of biologicalmaterial in MDs.

In the practice of this invention it is preferred to measure the changein the amount of biological material in MDs subsequent to one or moreincubations. More specifically, it is preferred to utilize MDs withindividual occupation such that the amount of biological materialassociated with individual biological entities, particularly cells, canbe measured prior to at least one incubation, and also subsequent atleast one incubation, so that the change in amount of biologicalmaterial is measured. Thus, it is particularly useful to carry out thepreceeding process wherein the change in amount of biological materialis used as an indication of viability of biological entities.

In cases in which the stringent criterion of biological entitiy growthis not required as the basis of determining viability, and thebiological entities consist of small multicellular organisms, groups ofcells, cells, protoplasts, vesicles, spores, organelles and parasites,it is possible to use this invention with short incubation, oressentialy no incubation, by using vital stains in combination with thevolume measurements of MDs. Vital stains respond to one or moreimportant biochemical or physical functions of biological entities,particularly cells, such that said functions can often be measured morerapidly than growth. Representative types of vital stains includetransmembrane potential stains, membrane exclusion stains andintracellular enzyme activity responsive stains. Specific vital stainsinclude cyanine dyes, propidium iodide, ethidium bromide, fluoresceindiacetate, carboxyfluorescein diacetate and fluorescein isothiocyanatediacetate. Thus, by exposing MDs to at least one vital stain andsubsequently measuring both the vital stain and volumes of theassociated MDs, an enumeration of viable biological entities can beobtained.

Although this invention can be used to obtain an approximate enumerationwithout statistical analysis applied to the MD measurements, the mostaccurate determinations involve statistical analysis which utilizes bothbiological material measurement and MD volume measurement. Suchstatistical analysis involves scoring each MD, or specimen of MDs, asoccupied or unoccupied. Additional information can be obtained byfurther scoring each MD according to the amount of biological material,so that growth of biological entities is measured and used as the basisfor determining viability. In the case that each MD, or specimen of MDs,is scored as occupied or unoccupied, the volume of the corresponding MD,or volume of the corresponding specimen of MDs, is utilized, such that astatistical frequency distribution of the occurence of occupation fordifferent ranges of MDs volumes, or MD specimen volumes, is determinedfrom the measurements, and this frequency distribution used to determinethe average number of viable biological entities per volume of samplewhich was used in the formation of MDs, and which therefore comprises aviable enumeration for the sample.

It is preferred to utilize Pisson statistics or modified Poissonstatistics with the measured frequency-of-occurrence of occupation inMDs within different MD volume ranges. As in the conventional, standardmethod of viable plating of cells, random mixing and the Poissonprobability distribution are used to obtain an enumeration. The use, ifnecessary, of iterative computations results in a self-consistentdetermination described by the Poisson probability function if thebiological entities were randomly distributed into MDs during the MDcreation process. An initial, trial value of ρ is used, and the initialoccupation distribution for the measured V_(MD) distribution iscomputed. The initial value of ρ is then adjusted, according to whetherthe computed distribution results in more or less occupation thanmeasured. This process is continued until there is agreement, preferablyto within 10 to 30%, but depending upon the application, and then thisvalue of ρ is corrected for dilution to obtain ρ_(s), which is thedesired viable cell enumeration, expressed as the number of viablebiological entities per volume of sample.

The average number of initial biological entities, n, in MDs within arange of volumes V_(MD) is related to the sample's cell concentration,ρ, through the relation ##EQU6## provides a determination of ρ_(s), theviable enumeration. Here f_(D) is the dilution factor defined byequation (3). The determination of n and V_(MD) for a statisticallysignificant number of occupied MDs allows ρ_(s) to be determined withsufficient accuracy, typically ±10%, to ±30%, which is better or aboutthe same as typical enumerations obtained by conventional viableplating. If desired, increased accuracy in ρ_(s) can be obtained bymaking and using measurements on a larger number of occupied andunoccupied MDs and/or groups of MDs.

Computation using suitable probability distributions such as thePoission probability formula, or modified Poisson statistics formulae,is also used, self-consistently, to identify which range of sizes withina MD specimen have a high probability of being unoccupied, individuallyoccupied or multiply occupied. Examples of suitable MD creationprocesses are described elsewhere in this disclosure. An advantage ofprocesses which produce a wide range of MD sizes is that a wide range ofsample cell concentrations, ρ, can be used. These conditions are usefulfor samples having a large specimen of MDs in which some significantfraction of the MDs will have volumes which correspond to having a highprobability of being unoccupied or individually occupied.

After formation, the MDs are placed in desired conditions fordetermining growth. Typically MDs can be suspended, or located, in amedium of a wide range of different compositions, and at different pH,temperature, partial pressures of oxygen and carbon dioxide, etc., sothat, as described elsewhere in this disclosure, growth under a widerange of growth medium conditions can be determined.

After an incubation period the MDs are exposed to one or more stainingprocesses, depending on the type of biological entity and the purpose ofthe growth determination. In the general case wherein growth analysiswithout further use of the biological entities is desired, any stainingprocess, including both those which kill biological entities and thosewhich do not kill entities, can be used. In the case wherein furtheranalysis or use of viable biological entities is desired, staining whichallows biological entitiy survival is used.

Following exposure of the MDs to suitable stains, MDs are individuallymeasured, or measured in small groups, provided that the probability offinding more than one biological entity-containing MD in the group issmall. In the exemplary case of cells stained by fluorescent compounds,optical analysis such as digital fluorescence microscopy or flowcytometry is used to analyze individual MDs, or groups of MDs, using awavelength band sufficiently different from that used for any detectionof measurement of MD properties. Thus, simultaneous, or serial,measurement of MD properties and of cells within said MDs is possible.The associated fluorescence signals are acquired and analyzed, withcorrection for spectral overlap if necessary, by conventional means.

The magnitude of the optical signal due to the biological entity stainin each MD, or MD group, is compared to the fluorescence of individualcells, whether or not such individual biological entities are entrappedin MDs, thereby providing a calibration. Comparison of the MD, or MDgroup, signal magnitude to that of individual biological entitiesprovides the basis for determination of growth of individual biologicalentities. For example, in the important case of cells, such comparisonof signal magnitude provides the basis for determination of growth ofindividual cells into microcolonies of two or more biological entities,which can be made within about one generation time, but without a needfor significant prior culture to obtain large numbers of cells, andprovides the basis for establishing that the occupied MDs contain viablecells, as determined by the requirement of growth from one into two ormore cells.

This invention can be used to obtain a viable enumeration of biologicalentities such as small multicellular organisms, groups of cells,individual cells, protoplasts, vesicles, spores, organelles, parasites,viruses, nucleic acid molecules, antibody molecules, and aggregates ofmolecules It is preferred to use this invention to enumerate cells suchas animal cells, plant cells, insect cells, bacterial cells, yeastcells, fungi cells and mold cells.

Representative suitable means for measuring biological material withinMDs, using naturally occuring properties of biological entities, orusing stains, includes physical means such as optical, weighing,sedimentation, field flow sedimentation fractionation, acoustic,magnetic, electrical and thermal means. It is preferred to use opticalmeasurements wherein biological material and gel microdroplet volumesare measured using optical phenomenona such as light scattering, lightabsorbance or colorimetric, fluorescence, time-delayed fluorescence,phosphorescence and chemiluminescence.

Optical measurements can be often enhanced by treating or exposing MDsto at least one staining process, wherein at least one stain is utilizedto enhance the measurement of biological material. Representativesuitable types of stains include stain indicative of biologicalcomposition, stain indicative of enzyme activity, and stain indicativeof cell membrane integrity Such stains are generally selected to havereadily measureable properties such as fluorescent stains, lightabsorbance stains and light scattering stains, and can be furtherselected according to the class of biological material which is stained,including, therefore, stains such as nucleic acids stains, proteinstains, lipid stains, cell membrane stains, cell wall stains, stainsresponsive to enzyme activity, stains responsive to transmembranepotentials and cell surface receptor stains.

It is also useful to practice this invention wherein optical measurementis made using apparatus such as flow cytometry apparatus,flow-through-microfluorimetry apparatus, optical particle analyzersapparatus, fluorescence microscopy apparatus, light microscopyapparatus, image analysis apparatus and video recording apparatus

Electrical measurements also have significant advantages, as electricalsignals can be coupled directly to computational means. Thus, it isuseful to practice this invention by employing electrical measurementmeans to measure biological material within MDs and also the volume ofMDs. Electrical measurements useful with GMDs include those involvingelectrical resistance particle analysis apparatus and dielectricproperty measurement apparatus, while those useful with LMDs involvesdielectric property measurement apparatus.

For example, it is well established that a resistive cell counter, oftentermed a Coulter Counter, can use electrical resistance measurement todetermine cell volume (see, for example, Kachel in Flow Cytometry andSorting, Melamed et al (Eds), Wiley, N.Y., pp. 61-104). In the case ofGMDs, the gel matrix of GMDs generally has a high molecular weightcutoff property, such that only large molecules are excluded, with theresult that the gel matrix offers only small electrical resistance ifGMDs are suspended in an aqueous medium comprising an aqueouselectrolyte with small ion composition similar to that of physiologicalsaline (about 0.9% NaCl). For this reason, GMDs without biologicalentities such as small multicellular organisms, cells, protoplasts,vesicles and spores have electrical resistance essentiallyindistinguishable from such aqueous electrolytes, and therefore are notelectrically measured, while cells contained with the GMDs are measured.Specifically, formation of microcolonies in GMDs leads to electricalresistance of the GMDs which increases with microcolony size, andthereby provides an electrical means for measuring the amount ofbiological material in GMDs.

The volume of the corresponding GMDs, or specimens of GMDs, can beobtained by using other means, including optical means responsive to thegel matrix of GMDs, or responsive to marker entities provides withinGMDs. Alternatively, by providing marker entities with measureableelectrical or magnetic properties in GMDs, it is possible to measure thevolume of the corresponding GMDs, or specimens of GMDs, by electrical ormagnetic means. For example, by providing marker entities comprisingparticles of a high dielectric constant such as barium titanate, it ispossible to measure the total amount of such dielectric material in eachGMD, or specimen of GMDs, and thereby to measure the volume of saidGMDs. A similar embodiment involves the use of marker entities withmeasureable magnetic properties, such as magnetite particles which havemeasureable magnetic properties which can be measured by well knownmeans such as positioning a coil in proximity to the orifice of theresistive particle counter. In these exemplary cases the electricalresistance of GMDs can be insignificantly altered by the presence of themarker entities, because even in the case that large numbers, e.g. 10⁵,of marker entities are used in a 50μ diameter GMD, the spacing of themarker entities within the gel matrix is sufficient so as to notsignificantly impede the movement of the small ions which predominantlydetermine the electrical resistance of such GMDs

Measurements on Mixed Biological Populations

Although many samples contain a single type of biological entitity, forexample, a monoculture of microorganisms wherein all of themicroorganisms are of the same type, a great many samples obtained inbiology and medicine are mixed populations, in that at least two typesof biological entities are present in the sample, generally such thatneither the relative numbers of the different types nor the absolutenumbers is known a priori. The present invention provides general meansfor measuring biological entities in mixed population samples, whilerequiring minimal or no pretreatment of the sample, and can yield suchmeasurements rapidly. The general process of this invention comprisesthe steps of: (a) creating microdroplets from a sample of the mixedpopulation, and (b) making at least one measurement which is sensitiveto at least one type of biological entity, and one additionalmeasurement which is sensitive to at least one other type of biologicalentity.

In this process it is preferred that there is a high probability thateach MD contains less than two types of biological entities, but theprocess can also be carried out under conditions in which there is ahigh probability that each MD contains at least one type of biologicalentity. In many cases, but not all, it is further desirable to providethe additional step of measuring MD volumes.

For example, formation of MDs which are LMDs from a sample containingtwo different types of microorganisms, I and II, with I havingsignificantly greater metabolic acid production and secretion rate thanII, when provided with the particular growth medium provided. Then, withor without dilution of the sample, by forming LMDs which have a range ofvolumes, V_(MD) =V_(LMD), or forming LMDs which have essentially thesame volume, LMDs can be formed which have a high probability of beingunoccupied or individually occupied, so that each occupied LMD has ahigh probability of containing one initial type I, or one initial typeII, microorganism. By providing optical pH indicators, either lightabsorbance or fluorescent, the LMDs can be incubated such that the acidproduction of the type I allows measurement and identification of theLMDs which contain that microorganism, thus providing the basis formeasurement of one type of biological entity in that mixed population.

In another example, if GMDs are formed from a suspension containing twodifferent types of cells, type A and type B, comprising the mixedbiological sample, and it is further assumed that: (a) type A growssignificantly faster than type B for the conditions provided, and (b)type A can be labeled with a Green Fluorescence labeled antibody to typeA surface antigens, and type B can be labeled with a Red Fluorescencelabeled antibody to type B surface antigen, then the growth of type Aand type B can be separately and simultaneously determined. Continuingthis example, a portion of the mixed population sample is converted intoGMDs, thereby incorporating cells of both types into GMDs. A specimen ofthe GMDs can be exposed, simultaneously or consecutively, to bothantibodies, such that antibodies enter the GMDs and bind to surfaceantigens, thereby labeling the amount of biological material, in thiscase amount of surface antigen, for both cell types. The amount of GreenFluorescence and Red Fluorescence in GMDs is then measured, to therebyprovide a measurement of the amount of biological material associatedwith type A and type B. Following incubation of at least one additionalspecimen of the GMDs at desired conditions, growth of both types ofcells is allowed to occur. All or a portion of this specimen can then beexposed to the same preparation of fluorescence-labeled antibodies,which provides a distinguishable measurent of the amount of biologicalmaterial associated with type A and type B. The measured amount of eachtype of biological material can then be quantitatively compared with anamount of biological material of each type in non-incubated GMDs,thereby providing a measurement of the growth of each type of cell inthe sample. As demonstrated by this example, GMDs are used to containbiological entities so that growth can be measured, but the volume ofthe GMDs need not necessarily be measured.

Although measurement of at least one type of biological entity of amixed population is often desired, it is possible to use the results ofsuch measurement process to further provide the basis of physicalisolation of MDs containing one type of biological entity. In thisfurther process, the value of at least one measurement is used toprovide the basis for applying at least one force to the correspondingMDs. This force results in physical manipulation of such MDs, and thenphysical isolation of such MDs. In this way MDs containing a first typeof biological material are isolated from MDs not containing the firsttype of biological material. Following such isolation of MDs thebiological entities contained within the MDs can be isolated or removedfrom the MDs by methods described elsewhere in this disclosure, therebyproviding isolation of the biological entities

Although measurements of a mixed population sample do not in all casesrequire measurement of the volume of the associated MDs, in most cases,the practice of this invention involves the measurement of MD volumesand the use of statistical analysis to determine the number ofbiological entities in MDs. In such cases, it is preferred to usestatistical formulae such as Poisson statistics and modified Poissonstatistics. These formulae provide a relation between the volume,V_(MD), the particular occupation or number, n, of initial biologicalentities in a MD, and the average number, n, of biological entities in aMD of this volume. Thus, by applying these formulae in an iterativefashion, which is generally well known, and which can readily byaccomplished using a computer, in the process of this invention, theprobability that a measured MD has individual occupation can bedetermined. By then further using measurements which can distinguish atleast two types of biological entities, it is possible to catagorize MDmeasurements into at least two catagories, and which, through the use ofthe statistical formulae, have a high probability of relating tomeasurements on only one type of biological entity.

Thus, to summarize this process, as a result of statistical anlysis onmeasurements which distinguish at least two types of biological entitiesand on MD volume measurements, the measurements which have a highprobability of relating to at least one type of biological entitity froma mixed population sample can be obtained. Such measurement results arepossible because the process of this invention provides a statistical,usually essentially random, separation of biological entities into MDs,such that the subsequent measurements and statistical analysis allowseparated measurements of biological entities. Thus, using this andsimilar versions of the method described herein, it is also possible tomake useful measurements on a mixed sample under conditions wheremeasured GMDs have a high probability of containing less than twobiological entities.

Although it is preferred to make measurements using MDs which areindividually occupied, it is possible to make useful measurements evenif MDs are multiply occupied. For example, as previously described, iftwo cell types, type A and type B, are measured by using antibodies forsurface antigens, and one antibody has a Green Fluorescence label andthe second a Red Fluorescence label, the growth of each type of cell canbe separately determined.

In another example, if the average occupation due to all types ofbiological entities of interest, n, is greater than about n=0.15, theprobability of initial occupation by an individual biological entitydecreases, such that if n>>0.15 most MDs are multiply occupied. Even inthe case of such multiple occupation, however, it is often possible tomake useful measurements on MDs formed from a mixed population. Forexample, if n=3, for a given size range of MDs, then as describedelsewhere in this disclosure, the probability of having n>7 is small, sothat there is a low probability of having more than 6 of one type in thepresence of the other type. In many cases the biological entities differsignificantly in properties such as biological material composition, orin growth, so that useful measurements can be made in such cases.However, as previously described, the preferred use of this invention isto utilize GMDs which have a high probability of containing at least onebiological entity.

The use of this invention with measurements which determine growth of atleast one type of biological entity is particularly useful, and can befurther extended by exposing at least one specimen of GMDs to conditionswhich affect the growth of at least one type of biological entity. Forexample, the use of selective growth media is well established inmicrobiology. The use of such media allows the outgrowth, typicallyaccomplished with long incubations which correspond to a large number ofdoubling times of the selected microorganisms in the presence of largenumbers of other types of microorganisms. Specifically themicroorganisms include those which do not grow, or grow much moreslowly, than the selected type. Identical or similar selective media canbe used with this invention, but it is not necessary to incubate forlong periods. In this case it is preferred, but not necessary, to usePoisson statistics or other suitable statistics to identify GMDs whichhave a high probability of individual occupation.

It is often useful to practice this invention by exposing microdropletsto conditions which affect the growth of at least one type of biologicalentity. For example, consider the illustration wherein the MDs are GMDs,and the provision of a selective medium results in zero growth of onetype of bacteria and growth of a second bacteria type such that thedoubling time for the growing type is t₂ =30 minutes, and that the lagtime for establishing growth is only a few minutes. In this case,following an incubation of approximately one hour the non-growingbacteria will still be present as single bacteria, such that GMDs withn=1 or individually occupied GMDs will have either single bacteria ifoccupied by the non-growing type, or will have microcolonies of 4bacteria if occupied by the growing type. Continuing this illustration,if each type of bacteria has essentially the same magnitude of RedFluorescence signal following the use of a propidium iodide stainingprotocol, the non-growing individual cells and microcolonies of 4 cellscan be readily distinguised. Individually occupied GMDs with growingbacteria will have Red Fluorescence signals about four times those ofindividually occupied GMDs with non-growing bacteria.

It is also useful to practice this invention by comparing the growth ofat least one type of biological material contained in microdroplets tothe growth of at least one other type of biological material containedwithin the microdroplets. For example, consider a related illustration,wherein again two types of bacteria comprise a mixed population, but nowwith the less optimal case wherein the selective conditions result innon-zero but different growth rates. Specifically, consider the casewherein type A has a doubling time, t_(A2) =30 minutes, and type B has adoubling time, t_(B2) =45 minutes. In such a case it is preferred, butnot necessary, to use Poisson statistics or other suitable statistics toidentify GMDs which have a high probability of individual occupation. Inthis case, assuming a lag time of only a few minutes, following anincubation of approximately 60 minutes, the type A bacteria will bepresent in individually occupied MDs as microcolonies of about 4 cells,cells, while the type B bacteria will be present as microcolonies ofabout 2.5 cells. Thus, individually occupied GMDs, i.e. GMDs initiallycontaining one cell, will have either microcolonies comprised of about 4cells if type A, or will have microcolonies of about 2.5 cells if typeB. Continuing this example, if each type of bacteria has essentially thesame magnitude of Red Fluorescence signal following the use of apropidium iodide staining protocol, the microcolonies of about 4 cellsand microcolonies of about 2.5 cells can be readily distinguised, as theRed Fluorescence signals will be proportional, in this example, tomicrocolony size. Using this type of measurement and analysis, theaverage growth rate of both types of cells can be simultaneouslydetermined from the mixed sample. This illustrates a basic method formaking rapid measurements on a mixed population. This example alsoserves to illustrate the use of this invention to use biological growthto distinguish at least two types of biological entities usingbiological growth Many variations of this example, includingstraightforward extension to more than two cell types, are possible.

A general version of this invention involves the use of measurement ofbiological material with sufficient specificity that at least one typeof biological material can be distinguished from at least one other typeof biological material. For example, fluorescence-labeled antibodies tosurface antigens provide a general basis for such specificity ofbiological material measurements. This has been described previously bymeans of an example based on a Green Fluorescence labeled antibody to afirst surface antigen, and a Red Fluorescence labeled antibody to asecond surface antigen, with growth of both cell types then possible.

The process of this invention further allows viable enumeration of oneor more biological entities of a mixed population sample. As illustratedpreviously, the growth of different types of biological entities can bedetermined separately and simultaneously. By further utilizing MD volumemeasurements with a statistical analysis based on Poisson statistics orother suitable statistics, the number of viable biological entities pervolume of sample can be obtained for one or more types of biologicalentities, which comprises obtaining a viable enumeration of at least onetype of biological entity

Differences in growth under selective medium conditions can be enhancedby exposing one or more specimens of GMDs to compounds whichsignificantly alter growth, such that one or more incubations of GMDsare provided with such compounds present. This allows two or more typesof biological entities to be measured by significantly altering thegrowth of at least one type of biological entity. Examples of suitablegrowth altering compounds include antibiotics, antimicrobial compounds,antifungal compounds, chemotherapeutic compounds, toxic compounds,cytotoxins, irreversible inhibitors, reversible inhibitors, mutageniccompounds, hazardous compounds, hormones, growth factors, growthenhancers, nutrients, vitamins, food preservatives, pesticides andinsecticides

In addition to making measurements of differential growth and/ormeasurements which measure different types of biological material, theprocess of this invention can be further extended by making measurementswhich are indicative of different types of biological entity function.More specifically, it is useful to make measurements wherein at leasttwo types of biological entities are determined by a measurementselected from the group consisting of biological material, biochemicalactivity, production of molecules, degradation of molecules, secretionof molecules, metabolism, membrane integrity, enzyme activity andgrowth. Thus, for example, if biological entities such as cells differin their ability to produce molecules, the resulting differences inproduction can be measured by using a stimulus such as electroporationto release molecules for capture at binding sites and subsequentmeasurement. Finally, if biological entities such as cells differ intheir enzyme activity when exposed to certain conditions, and theresultant differing enzyme activity is measured, this type ofmeasurement also distinguishes types of biological entities.

As described previously, optical means are preferred for measurement ofbiological material and microdroplet volumes, using optical phenomenasuch as light scattering, light absorbance or colorimetric,fluorescence, time-delayed fluorescence, phosphorescence andchemiluminescence. It is also often useful to incorporate markerentities into GMDs in order to enhance microdroplet volume measurement,using marker entities such as beads, non-biological particles, crystals,nonaqueous fluid inclusions, viable cells, dead cells, inactive cells,virus, spores, protoplasts, vesicles, stains and dyes. Alternatively, itis possible to pretreat GMDs so as to attach marker entities to at leastone gel matrix constituent. Furthermore, marker entities can beincorporated into microdroplets after creation of the microdroplets.

Alternative measurement means include optical, weighing, sedimentation,field flow sedimentation fractionation, acoustic, magnetic, electricaland thermal means. Further, as described elsewhere in this disclosure,it is useful to use optical measurements are selected from the groupconsisting of light scattering, light absorbance or colorimetric,fluorescence, time-delayed fluorescence, phosphorescence andchemiluminescence This process of this invention can also involve theadditional step of exposing microdroplets to at least one compound whichaffects biological material prior to measurement, thereby enhancing thedistinction between at least two types of biological entities.

This invention can be used with mixed popultions in order to makemeasurements of biological entities such as small multicellularorganisms, groups of cells, individual cells, protoplasts, vesicles,spores, organelles, parasites, viruses, nucleic acid molecules, antibodymolecules, antigen molecules, and aggregates of molecules, and it ispreferred to make measurements on cells such as animal cells, plantcells, insect cells, bacterial cells, yeast cells, fungi cells and moldcells, and also cells such as normal human cells, human cancer cells,pathogenic bacteria, pathogenic yeast, mycoplasms, parasites, andpathogenic viruses.

Furthermore, although this invention can be carried out by measuring theamount of biological material in at least one gel microdroplet bymeasuring a naturally occuring optical signal associated with thebiological material, such as optical signals selected from the groupconsisting of light scattering, light absorbance or colorimetric,fluorescence, time-delayed fluorescence, phosphorescence andchemiluminescence, it is preferred to utilize at least one stainingprocess involving at least one stain for biological material.Representative types of stains which can be used are stains such asstain indicative of biological composition, stain indicative of enzymeactivity, and stain indicative of cell membrane integrity.

It is also useful to practice this invention in an embodiment wherein atleast one stain is used for biological entity identification and atleast one stain or a naturally occuring optical signal is used todetermine biological entity growth. A particularly useful generalversion of this embodiment involves the use of at least onefluorescence-labeled antibody is used for biological entityidentification.

Thus, this invention can be used to make a variety of measurements onmixed biological populations which cannot be readily made by priormethods.

Provision of External Influence on Biological Entities

In many tests and assays the interaction of biological entities withexternal sources of chemicals, biological factors or physical fields areextremely important, and it is highly desirable to provide means forextending MD measurement and isolation processes to include the effectsof such influence. In order to provide such external influence onbiological entities, the biological entities are incorporated into MDs,so the MDs which contain biological entities can be introduced intoposition of proximity to a source, maintained in position in proximityto a source or removed from proximity to a source. In the generalpractice of this invention, sources of influence include biologicalsources of influence, chemical sources of influence, physical sources ofinfluence, and combinations of biological, chemical and physical sourcesof influence are provided by using MDs,

In general, however, it is often preferred to provide influence byutilizing MDs which are GMDs, particularly GMDs which are surrounded byan aqueous medium such that the aqueous medium contacts the aqueousinterior medium of GMDs, thereby allowing chemical and small biologicalentities to be exchanged between the GMDs and the aqueous medium. It isalso possible to provide influence by using LMDs and/or GMDs surroundedby a non-aqueous fluid, as most types of physical influence is readilyprovided , but the chemicals which can be exchanged is more limited, andrelatively few biological entities can be exchanged. For aqueoussurrounded GMDs, exemplary surrounding aqueous media include aqueousgrowth medium, physiological fluids, human body fluids, animal bodyfluids, organ perfusates, suspensions of cells, suspension of smallmulticellular organisms, animal tissue homogenates, plant tissuehomogenates, cell culture medium, culture medium for microorganismscontaining biological material, defined culture medium formicroorganisms, defined culture medium for mammalian cells, blood, bloodplasma, urine, cerebral spinal fluid and interstitial fluid. Uponexposure of GMDs to one or more such aqueous media or environments, anddepending on the molecular filteration characteristics of the gelmatrix, most chemical compounds and some small biological entities canenter the GMD by partitioning into the aqueous environment of the gelmatrix. These can be transported, often by diffusion, but also by driftand/or convection, within the GMD, thereby exposing biological entitiescontained within GMDs to chemical compounds and/or small biologicalentities contained within the aqueous environment.

In the case of such GMDs, the present invention provides means forinfluencing biological entities to external influence thought thegeneral means of providing physical forces which allow manipulation ofGMDs, such that GMDs can be moved into and within an aqueousenvironment. Such manipulation of GMDs allows GMDs to be positioned inproximity to sources of influence which are external to GMDs, such thatchemical compounds of the aqueous environment in proximity to one ormore external sources can enter GMDs and thereby interact withbiological entities within GMDs, and thus provide influence on thebiological entites. More specifically, this process provides a generalmeans for providing external influence on biological entites byutilizing physical manipulation of GMDs, containing at least onebiological entity, so as to affect the proximity of at least one GMD toan influence source which is external to the GMDs. Thus, it isparticulalry useful to provide influence on biological entities byutilizing GMDs in an aqueous fluid.

However, because of the greater generality of this invention, it isappropriate to describe most of this invention in terms of the use ofthe more general case of MDs surrounded by either aqueous fluids ornon-aqueous fluids. Following the provision of influence, it isgenerally useful to determine the effect of the influence on biologicalproperies of biological entities contained within the MD. This isparticularly relevant for biological properties such as biologicalmaterial, biochemical activity, production of molecules, degradation ofmolecules, secretion of molecules, metabolism, membrane integrity,enzyme activity and growth, as these are directly or indirectly involvedin many assays and tests In order to flexibly provide influence due toone or more biological sources it is useful to provide such biologicalinfluence by selecting a source from the group consisting of cells,feeder cells, animal tissues, plant tissues, animal tissue homogenates,plant tissue homogenates, perfused organs, whole animals, plants, cellsuspensions, biological entities within gel microdroplets, cellcultures, body fluids.

As used herein, the term biological influence describes the influenceassociated with chemical compounds and small biological agents such asphage, which have biological origin and can be present in the aqueousenvironment in proximity to a biological source. Representativebiological sources include cells, feeder cells, animal tissues, planttissues, animal tissue homogenates, plant tissue homogenates, perfusedorgans, whole animals, plants cell suspensions, biological entitieswithin gel microdroplets, cell cultures, body fluids. Chemical sourcesinclude any entity which provides to an aqueous environment one or morechemical compounds, and can therefore include biological sources.Physical sources include any entity which is the source of physicalconditions in an aqueous environment. These include physical processessuch heat, electrical energy, magnetic energy, optical energy,radioactivity or ionizing radiation, and acoustic energy which alterchemical composition of chemical compounds in an aqueous environment.Physical sources also include any entity which directly provides heat,electrical energy, magnetic energy, optical energy, radioactivity oracoustic energy to MDs, thereby providing influence on biologicalentities contained within MDs.

By using the process of this invention in which MDs can be manipulated,including specifically manipulation which results in MDs being near,maintained near and removed from one or more sources of externalinfluence, external influence can be caused on biological entities suchas small multicellular organisms, groups of cells, individual cells,protoplasts, vesicles, spores, organelles, parasites, viruses, nucleicacid molecules, antibody molecules, antigen molecules, and aggregates ofmolecules It is particularly useful to provide influence on cells suchas animal cells, plant cells, insect cells, bacterial cells, yeastcells, fungi cells and mold cells, and such as normal human cells, humancancer cells, pathogenic bacteria, pathogenic yeast, mycoplasms,parasites, and pathogenic viruses In the important case of mammaliancells it is preferred to provide influence on cells such as of normalhuman cells, cancerous human cells and hybridoma cells.

In order to provide complex biological influence, wherein many differentand complex biochemical pathways may be involved, and wherein differenttissues may participate, it is possible to reversibly position MDs,particularly GMDs, within whole animals such as mouse, immunodeficientmouse, rat, rabbit, primate, goat, dog, horse, pig, guinea pig and andhuman being. These are important biological sources of externalinfluence, and additional related sources comprise one or more perfusedorgans from such animals Thus, for example, GMDs containing magneticforce-coupling entities in addition to biological entities can beinserted into an animal, so that complex biological influence can beprovided. If desired, the biological entities within the GMDs can beprotected from an immune response by the animal by providing anappropriate molecular weight cutoff property for the gel matrix of theGMDs. An exemplary means for accomplishing this protection is to usecomposite GMDs wherein an outermost gel region surrounding one or morebiological entity-containing regions is used with a gel with amoderately low molecular weight cutoff.

Additional flexibility can be provided by supplying influence due to oneor more sources which are themselves contained within MDs, and in thisgeneral case it is the relative position of the influencing MDs and theinfluenced MDs which is important. Thus either or both types of MDs canbe manipulated The flexibility of this invention can also be increasedby providing and utilizing gel material for MDs which are GMDs whereinthe gel matrix which allows selective passage of molecules.

In order to physically manipulate MDs with respect to one or moresources of influence, the process of this invention provides physicalforces such as, wherein the means for generation of such forces iswell-known or is described elsewhere in this disclosure In order to moreeffectively provide such forces it is useful to supply one or more typesof coupling entities, or force-coupling entities, wherein such entitiesare selected from the group consisting of beads, non-biologicalparticles, bubbles, and non-aqueous fluid inclusions with force couplingproperties selected from the group consisting of ferromagneticproperties, diamagnetic properties, paramagnetic properties, dielectricproperties, electrical charge properties, electrophoresis properties,mass density properties, and optical pressure properties It is generallypreferred to provide magnetic force by incorporating magnetic couplingentities into MDs, and an exemplary type of magnetic force couplingentities are those comprised of magnetite.

Forces for MD manipulation near sources of influence can also beprovided by incorporating coupling entities with a mass densitydifferent than the gel density, so that physical forces such as fieldflow sedimentation fractionation force, acoustic force, gravitationalforce, centripetal force, centrifugal force and non-rotationalacceleration force can be utilized.

An illustration of a source of physical influence relates tomanipulations and measurements involving the use of heat and ionizingradiation for treatment of cancer, wherein cancer cells can be killed byapplication of heat and/or ionizing radiation. The use of ionizingradiation is well established as a therapeutic modality for thetreatment of cancer, and the use of elevated temperature, orhyperthermia, either alone or in combination with ionizing radiation,has been more recently established as a therapeutic modality. In suchtreatments, a general tendency of increased cancer cell killing relativeto normal cell killing is exploited. In order to carry out in vitrotests of the sensititivy, and the variability of sensitivity, of cancercells to heat and/or ionizing radiation, cancer cells can beincorporated into GMDs. The GMDs are then exposed to the physicalinfluence comprising elevated temperature and/or exposure to ionizingradiation, and then, if desired, incubated. The MDs are then measuredfor changes in the amount of biological material or biochemicalactivity. It is preferred to measure the amount of biological materialrelative to one or more controls, thereby providing a measure of growthof the cells exposed to physical influence relative to the growth ofcontrol cells in which are not exposed to the physical influence.Further, staining protocols can be used which test for membraneintegrity. Thus, the fluorescent stain propidium iodide, and additionalor separate staining with vital stains such as FITC diacetate can beused as the basis of a short term indicator of viability.

The process of this invention relating to provision of physicalinfluence can also be carried out under conditions that approximate invivo conditions. For example, GMDs containing cells to be tested can beplaced within the body of an animal, such as a immunocompromised mouse,and the animal then exposed to conditions which approximate hyperthermiaand/or ionizing radiation treatment in animal species, including humans.This provides external influence on the cells which is a combination ofphysical influence relating to heat and/or ionizing radiation, and alsoexternal biological influence relating to the biochemical environmentwithin the so treated animal. Following such provision of externalinfluence on cells, measurements such as those relating to cell death,cell survival, cell growth and biochemical activity can be carried outusing aspects of this invention described elsewhere in the presentdisclosure. Thus, exposure to heat influence can be provided andutilized to determine cell death and cell growth under conditionsrelating to cancer hyperthermia treatment.

Similarly, the effect of the physical influence of ionizing radiation onbiological entities, particularly normal mammalian cells and cancerousmammalian cells, can be tested by the process of this invention, eitherunder completely in vitro conditions, or, by inserting MDs, particuarlyGMDs, into a test animal, or even a human being, under essentially invivo conditions. Thus, this invention can be used to determine celldeath and cell growth under conditions relating to cancer radiationtreatment.

The process of this invention can also be applied to combinations ofcancer treatment which include sequential or simultaneous combinationsof physical treatment such as ionizing radiation and hyperthermia, andchemical treatment, including established chemotherapy and newer therapybased on monoclonal antibodies and the like.

In the preferred embodiment of this invention, one or more sources ofexternal influence are selected from the group consisting of mammaliancells, yeast cells and bacterial cells. In the case of mammalian cellculture, biological entities such as cultured mammalian cells can beexposed to "feeder cells". These "feeder cells" comprise the externalsource of influence, which in this case, are compounds which enhance thegrowth of cells. In addition, it is often preferred to influencebiological entities, particularly cells, to the complex biochemicalinfluence which is be provided by whole animals, especially animalsselected from the group consisting of mouse, immunodeficient mouse, rat,rabbit, primate, goat, dog, horse and and human being. For example, thecomplex metabolic processes of whole animals can provide activation anddegredation of chemical compounds which is generally difficult toduplicate in cell culture. Thus, the use of the present inventionprovides a general means for reversibly exposing biological entitiessuch as cells to complex external influence resulting from whole animalactivation and metabolism of chemical compounds.

In the preferred embodiment of this invention, biological entities areselected from the group of bacterial cells, yeast cells and mammaliancells, particularly normal human cells, cancerous human cells andhybridoma cells.

This invention can also be used in a version wherein one or more sourcesof external influence are incorporated into microdroplets, therebyproviding a means for influencing biological entities wherein the sourceof influence can be manipulated by altering the position of the sourcewith respect to the influenced biological entities which are in otherMDs In this general case, different types of force can be used with theinfluencing MDs, for example magnetic force, while another force, forexample, a force depenedent on the mass density of MDs, can be used withthe influenced MDs Finally, in a related embodiment MDs sources ofinfluence can be incorporated into MDs, and the resulting MDsmanipulated so as to provide influence on biological entities which arenot contained in MDs For example, feeder cells can be incorporated intoGMDs with are also provided with magnetic force coupling entities suchas magnetite, so that the feeder cells can be positioned in proximity tocultured cells which are not in GMDs, and, following any desiredincubation, the GMDs containing the feeder cells can be readily removed.

Forces suitable for manipulation of MDs surrounded by a non-aqueousfluid are described elsewhere in this disclosure. Any of a variety ofphysical forces can be used to introduce GMDs surrounded by an aqueousfluid into close proximity to a source of influence, maintain GMDs inclose proximity to a source of influence, and to remove GMDs from closeproximity to a source of influence. Generally, the physical force isselected from the group consisting of electrical force, magnetic force,field flow sedimentation fractionation force, acoustic force, opticalpressure force, gravitational force, sedimentation force, non-rotationalacceleration force, centrifugal force and centripetal force.

Electrical force can be provided by interaction of an applied electricfield, including an electric field with field gradients, with dielectricparticles provided within the gel matrix of GMDs, or more generally byinteraction with charge groups associated with the gel matrix orcoupling entities provided within GMDs. Gravitational force,non-rotational force, centripetal force or centrifugal force andsedimentation force can all be applied by utilizing gel matrixcomposition having different mass density than the surrounding aqueousmedium and providing the corresponding physical force field oracceleration. Magnetic force can by applied by providing couplingentities within GMDs, said coupling entities having diamagnetic,paramagnetic or ferromagnetic properties different from the aqueousmedium which surrounds the GMDs. Representative coupling entities forapplying a magnetic force are magnetic particles, magnetic granuals,ferrofluid inclusions and the like. A preferred embodiment comprisesmagnetite (Fe₂ O₄) particles within GMDs. Alternatively, magnetic forcecan be applied directly in those cases wherein the diamagnetic,paramagnetic, ferromagnetic or electrical conductivity properties ofGMDs differs from the diamagnetic, paramagnetic, ferromagnetic orelectrical conductivity properties of the aqueous medium which surroundsthe GMDs. Acoustic forces can be applied by applying sound or acousticfields which preferentially interacts with the gel matrix of GMDs, orwith coupling entities contained within GMDs, such that the inclusion ofsaid coupling entities results in GMDs having a different mass density,or different mechanical compliance, than the aqueous medium whichsurrounds the GMDs. Optical pressure force can be applied by utilizingoptical radiation which interacts with the gel matrix or couplingentities contained within the GMDs, or with the larger biologicalentities contained within the GMDs (see, for example, Ashkin et al,Nature 330: 769-771, 1987). Sedimentation field flow fractionation forcecan be provided by simultaneously utilizing a hydrodynamic force and asedimentation force (see, for example, Levy and Fox, Biotech. Lab.6:14-21, 1988), and can be used to separate GMDs.

After one or more exposures to one or more compounds, and following oneor more incubation periods with or without controls, the MDs, that isLMDs and GMDs, are exposed to one or more staining processes, which aredescribed elsewhere in this application, but, as also describedelsewhere, if naturally occuring signals are adquate, the stainingprocesses are omitted.

Following exposure of the GMDs to suitable stains, individual GMDs areindividually measured, or measured in small groups, provided that theprobability of finding more than one cell-containing GMD in the group issmall. In the exemplary case of cells stained by fluorescent compounds,optical analysis such as digital fluorescence microscopy or flowcytometry is used to analyze individual GMDs, using a wavelength bandsufficiently different from that used for any detection of measurementof GMD properties so that simultaneous, or serial, measurement of GMDproperties and of cells with said GMD are possible. The associatedfluorescence signals are acquired and analyzed, with correction forspectral overlap if necessary, by conventional means.

The magnitude of the optical signal due to the cell stain in each GMD,or small group of GMDs, is compared to the fluorescence of individualcells, whether or not such individual cells are entrapped in GMDs,thereby providing a calibration. Comparison of the GMD signal magnitudeto that of individual cells provides the basis for determination ofgrowth of individual cells, for which the growth determination can oftenbe made within about one generation time, but without a need forsignificant prior culture to obtain large numbers of cells, and growthcan also be determined over several generations if desired.

By making a large number of such individual cell growth determinations,the distribution of growth rate, distribution of lag time, and theplating efficiency caused by the exposure to one or more compounds oragents can be automatically determined by computer calculation. Othermeasurements relating to cell survival and cell death, particularlyvital stains such as transmembrane potential stains, membrane exclusionstains and intracellular enzyme activity responsive stains, can also beused. Manual or visual inspection and scoring of GMDs can also be used,but is relatively labor intensive and therefore more prone to error.Thus, the preferred process is that conducted using the automatedmeasurement means.

Similarly, measurements, assays, tests and isolation procedures directedtowards the use of biological entities for the production of desirablecompounds, or the use of biological entities to provide processesdirected towards the degredation or modification of undesirablecompounds, such as toxic wastes, can benefit from biological orbiochemical activation of the biological entities, which activationcomprises a form of influence on the biological entities.

Homogeneous Specific Binding Assay

This invention can be used to provide measurement of certain types ofbiological entities, herein refered to as analyte entities, capable ofreacting with and binding two or more labled specific binding molecules,wherein the labeled specific binding molecules are measured directly bymeasuring one or more labels which have been attached to the individuallabeled specific binding molecules, or are measured indirectly throughthe subsequent binding of additional, labeling molecules which can bindto, and thereby label, the labeled specific binding molecule. Examplesof suitable specific binding molecules are antibodies, antigens, nucleicacids, avidin-biotin, enzyme inhibitors and lectins A key property ofanalyte entities is that the analyte entities have two or more specificbinding sites which can bind labeled specific binding molecules duringthe time required to from MDs from a sample containing the labeledspecific binding molecules.

Although the process of this invention is general, applicable to manytypes of specific binding molecules, this invention is most readilydescribed or illustrated in terms of its application to immunoassays.For example, the process of this invention is illustrated by consideringa analyte entity with two distinct epitopes. In the case of labeledlabeled specific binding molecules, a sample containing the analyteentity is exposed to two different labeled antibodies, with one antibodyspecific for each of the two distinct epitopes. Following mixing, ifdesired, and after waiting for diffusion, encounter and binding of thelabled antibody molecules, the analyte entities have a high probabilityof being specifically labeled with two labels because of the binding ofthe two labeled antibodies.

MDs are then formed, using methods described elsewhere in thisdisclosure, such that at least some of the MDs have a high probabilityof being individually occupied by the so labeled analyte entities. Oneor more measurements of the amount of label in each MD is then made,such that the measurement is capable of resolving the difference of onelabel from two labels, and other measurements are made which allow themeasurement of each MD volume, or the volume of each group of MDs It ispreferred to measure individual MDs for volume and enzyme activity, butin some cases two or more MDs can be measured together Such measurementis used to characterize each MD according to the number of labelscontained, for example 0, 1, 2 or more than 2 labels. Statisticalanalysis, such as that based on Poisson statistics, is then employedwith the measured frequency-of-occurence distribution for n_(F) =0 andn_(F) =1 to predict the number of MDs in each volume range which shouldhave n_(F) =2 because of a random distribution.

This random prediction is then compared to the measuredfrequency-of-occurrence, and the excess over random is attributed to thebinding of two labeled antibodies to the analyte entity. This excessfrequency-of-occurrence is then used with statistical analysis tocompute the concentration of analyte entities in the solution orsuspension from which MDs were formed, and is further corrected bycomputation, if necessary, for any dilution that was made whilepreparing the solution or suspension, thereby measuring theconcentration of the analyte entities in the sample.

Although a variety of suitable V_(MD) measurement processes aredescribed elsewhere in this disclosure, in some cases it is useful tomeasure one or more positive optical signals associated with moleculescontained within microdroplets and/or one or more negative opticalsignals associated with molecules contained in a fluid surroundingmicrodroplets, such as has been partially described previously as"negative fluorescence" (Gray et al (Cytometry 3: 428-434, 1983)

In order to describe this invention we use the following notation.

An: Subscript denoting analyte (molecule, virus, cell, etc.)

BS: Subscript denoting binding sites on the analyte.

F: Subscript denoting free or unbound LSBMs (Labeled Specific BindingMolecules)

L: Subscript denoting label of LSBMs

n_(An) : Particular number of analyte entities in a MD.

n_(An) : Average or mean number of analyte entities in a MD.

LSBM: Labeled specific binding molecule (e.g. labeled antibody).

n_(F) : Particular number of unbound LSBMs in a MD.

n_(F) : Average or mean number of unbound LSBMs in a MD.

N_(BS) : Number of binding sites on analyte capable of binding LSBMs.

n_(L),total : Total number of labels found within a MD (=n_(F) +N_(BS)n_(An))

n_(L),total : Average or mean number of labels found within a MD.

S: Signal obtained from a particular MD.

A property of many solutions and suspensions is that LSBMs and analyteentities are distributed randomly within the solutions and suspensionsif the LSBMs and analyte entities are free. As used herein, the termfree means that the LSBMs and analyte entities are not bound, andincludes the absence of binding of LSBMs to analyte entities. For thegeneral case in which the formation of MDs divides the solution orsuspension from which MDs are formed randomly into the small volumes ofMDs, the probability of unbound or free LSMBs occupying MDs is welldescribed by the Poisson formula.

More specifically, if unbound LSBMs are randomly distributed, theprobability of finding exactly n_(F) unbound LSBMs in any MD is,##EQU7## where the mean occupation is n_(F), n_(F) =V_(MD)ρF with V_(MD)a microdroplet volume, and ρ_(F) is the concentration of all the free orunbound labeled specific binding molecules in the general case whereinN_(BS) different binding sites (e.g. epitopes) are exploited, N_(BS)different LSBMs are used at the same concentration, and therefore theconcentration of unbound label in a MD with no analyte entitity isN_(BS)ρF.

Likewise, for randomly distributed analyte entities ##EQU8## so thatn_(An) =V_(MD)ρAn where microdroplet volume, and .sub.ρAn is theconcentration of the analyte. The variable n_(An) is random for a wellmixed system, and is therefore independent of n_(F).

The total number of measureable labels remains constant, unless one ormore degradative reactions occur, thereby modifying labels so as toeffect the measureable properties of the labels. Further, it is wellknown that non-specific binding can sometimes occur if macroscopic solidsurfaces or microscopic entities such as certain cell surfaces, virussurfaces and/or molecules are present. Such undesirable effects can alsodecrease the amount of free label. Generally, however, the combinationof label degradation and non-specific binding of LSBMs can be made smallby avoiding the use of macroscopic solid surfaces, since the occurrenceof label degradation is relatively rare. Thus, although a generalcondition is that total amount of LSBM is conserved. In many casesessentially all of the LSBMs exist free in solution or are specificallybound to analyte entities.

Although it is not generally necessary, it is often preferred to carryout an additional step following the reaction of LSBMs with analyteentities. This additional step comprises reducing the concentration ofthe free LSBMs, so that the concentration of free LSBMs relative to theconcentration of LSBM-analyte is reduced prior to formation of MDs. Inthis way, a relatively high concentration of LSBMs can be first employedin order to more rapidly drive the specific binding reactions which leadto the LSBM-analyte entity complexes

Following addition and mixing of LSBMs to a sample solution orsuspension, and a subsequent incubation during which LSBMs are allowedto bind to analyte entities, it is often desirable to reduce theconcentration of the remaining unbound or free LSBMs. This can beaccomplished by the additional step of adding purging entities, suchthat free LSBM encounters and binds to the purging entities. Suitablepurging entities include beads, non-biological particles, crystals,nonaqueous fluid inclusions, viable cells, dead cells, inactive cells,virus, spores, protoplasts and vesicles, which have surfaces withtightly bound molecules capable of tightly binding labeled specificbinding molecules. By using one or more additional signals (e.g. lightscattering, fluorescence) to distinguish the purging entities, the labelsignals associated with the purging entities can be ignored duringanalysis of the measurements. Alternatively, if the purging entitiesbind significantly larger numbers of label than analyte, the analysis ofmeasurements can distinguish such larger numbers of labels, and will notconfuse such labels associated with LSBM-analyte complexes.

A general attribute of the process of this invention is that it is notnecessary to accurately know the concentration of the LSBMs, since theconcentration of LSBM-analyte complexes is the information sought. Morespecifically, although the provided concentration of the LSBMs isreadily known, the concentration following reaction with and binding toanalyte is generally not known. This is further less known following theuse of any purging entities. However, it is often desirable to know thatconcentration of label in order to provide corrections to themeasurements. An advantage of this invention is that thefrequencies-of-occurrence of label is determined using many MDs, oftenof significantly different volumes, V_(MD), such that subsequentanalysis of such frequencies-of-occurrence allows computation of.sub.ρLSBM.

The process of this invention is illustrated by considering MDs with arange of volumes from V_(MD) to V_(MD) +ΔV_(MD), such that followingexposure of the sample to LSBMs and the subsequent step, if desired, ofadding purging entities, this range is characterized by n_(F) =0.15,n_(An) =0.15 and N_(BS) =2. In this case the Poisson formula predictsthe following probabilities.

    ______________________________________                                        n.sub.F P(n.sub.F,- n.sub.F = 0.15)                                                               n.sub.An  P(n.sub.An,- n.sub.An = 0.15)                   ______________________________________                                        0       0.8607      0         0.8607                                          1       0.1291      1         0.1291                                          2       0.00968     2         0.00968                                         3       0.000484    3         0.000484                                        ______________________________________                                    

In this illustration a MD with zero analyte thus has a probability ofabout 0.0097 of having two labels through random occupation by LSBMs,and the same volume MD has a probability of 0.1291 of having two labelsthrough occupation by one analyte entity. The excess probability of twolabels resulting from LSBM-analyte occupation compared to randomoccupation by two free LSBMs is

    ΔP=P(n.sub.An =2,n.sub.An =0.15)-P(n.sub.F =2,n.sub.F =0.15)=0.119(11)

Thus, if a sufficiently large number of occupied MDs are measured, thedifference in probabilities can be measured by measuring thecorresponding difference in frequency-of-occurrence of the measured andthat predicted based on the measured frequency-of-occurrence of n_(F) =0and n_(F) =1.

Continuing this illustration, the Poisson probabilities for n_(F) =0,n_(F) =1 and n_(F) =2 are

    P(n.sub.F =0,n.sub.F)=e.sup.-n.sub.F                       (12)

    P(n.sub.F =1,n.sub.F)=ne.sup.-n.sbsp.F                     (13)

    P(n.sub.F =2,n.sub.F)=1/2(n.sup.2 e.sup.-n.sbsp.F          (14)

The probability of measuring two labels resulting from random occupationis thus related to the probabilities for measuring zero randomlyoccuring label, and for measuring one randomly occuring label throughthe equation ##EQU9## The mean or average occupation, n_(F), of randomlyoccuring label can be obtained from the mathematical relation ##EQU10##The experimentally measured frequency-of-occurrence of n_(F) =0, n_(F)=1 and n_(F) =2 can now be quantitatively compared with theprobabilities obtained by computation using the Poisson probabilities.More specifically, the experimentally measured frequency-of-occurrence,f(n_(F)) and the Poisson formula are utilized to compute the differencebetween measured frequency-of-occurrence and the theoretical randomfrequency-of-occurrence. This can be used to interpret the difference,if statistically significant, to the occurrence of MDs with complexes ofanalyte and N_(BS) LSBMs. This is accomplished by measuring thefrequencies-of-occurrence and then computing first the mean value n_(F)associated with random occurrence, i.e. ##EQU11## and then using this incomputing the difference ##EQU12## This measured difference is used withthe total volume, V_(MD),total, of measured MDs in the volume rangeΔV_(MD) to compute the concentration of analyte. More specifically,##EQU13## wherein N MDs are measured within the volume range V_(MD) toV_(MD) +ΔV_(MD). Continuing this illustration further, the analyteconcentration, ρ_(An), is computed from ##EQU14## and thereby achievesthe desired result of measuring the analyte concentration.

Continuing this illustration still further, the error in the measurementof ρ_(An) can be estimated by using well-known methods of erroranalysis. As shown above, the determination of ρ_(An) depends onmeasured frequencies-of-occurrence and measured MD volumes. Thus, theerror in determining ρ_(An) can be computed using well-knownpropagation-of-error methods, so that the overall result of the processof this invention is a measurement of ρ_(An), and also a determinationof the error in the measurement.

More generally the process of this invention utilizes measurement offrequencies-of-occurrence in MDs, and also measurement of MD volumes,such that comparison between random occupation of MDs by label andnon-random occupation of MDs by label can be made. One general process,useful in cases wherein the average occupation by free label, n_(F), isless than one, utilizes the mathematical recursion relation for thePoisson formula, here used with the abbreviated notationP(n).tbd.P(n,n), so that the recursion relation is ##EQU15## so that onaverage the measured frequency-of-occurrences for random events will berelated by ##EQU16## and can be straightforwardly used to compute theexcess over random for occupation of MDs by label over a wider range ofoccupation.

An alternative method for providing statistical analysis of the MDmeasurement is useful in the case wherein significant measurement error,δƒ(n_(F)), in the frequency-of-occurrence is expected. This alternativemethod utilizes well-known averaging methods, which reduce error. Theoccurrence of free LSBMs and analyte entities within a MD arestatistically independent, so that the probability of having aparticular combination of n_(F) free label or free LSBMs and n_(An)analyte entities such that the total amount or number of labels is n_(L)=n_(F) +N_(BS) n_(An). This emphasizes that different combinations ofn_(L) and n_(An) can result in the same n_(F), and indicates that inorder to compute the probability of a given value of n_(L) a summationover the different possibilities giving the same n_(L) is needed.

A basic property of this approach is the recognition that n_(L) is alinear combination of n_(F) and n_(An), and that therefore theprobability function for n_(F) is given by the convolution of P_(F)(n_(F),n_(F)) and P_(An) (n_(An),n_(An)). Thus, using well-known resultsof fundamental probability theory, the average values n_(L), n_(F) andn_(An) are related by

    n.sub.L =n.sub.F +N.sub.BS n.sub.An                        (23)

and the variances of these same three parameters are related by##EQU17## where the variance is a measure of the spread or statisticalerror which is expected. By using the above two mathematical relations,and measurements of frequencies-of-occurrence to obtain determinationsof n_(L) and var(n_(L)), the parameter n_(An) can be computed. Thiscomputed value is used in Poisson statistics formulae with measuredvalues of V_(MD) to compute .sub.ρAn.

The statistical error in the average and the variance is wellcharacterized according to fundamental probability theory, and can beused with propagation-of-error analysis to compute the error in.sub.ρAn.

Both monoclonal and polyclonal antibodies with labels can be used asLSBMs in this invention. If monoclonal antibodies are used, N_(BS) isknown exactly, as is the average number of labels per LSBM. However, inthe related case wherein LSBMs are polyclonal antibodies, somewhatdifferent numbers of antibodies may bind, with varying avidities, toindividual analyte entities. such binding by different numbers, however,can generally be characterized by an average or mean number of bindingsites, N_(BS). The value of N_(BS) is determined from initial,calibrating experiments, as it is recognized that there may be somevariation in the actual number of bound LSBMS, that is, there may be adistribution, N_(BS) =N_(BS) ±δN_(BS) around N_(BS). In this case,measurement of frequencies-of-occurrence results in a superposition of(1) a random distribution, consistent with the Poisson formula for freelabel, and (2) a peaked distribution located at N_(BS) with widthδN_(BS).

A general computational process can be utilized with the measurements ofthe frequencies-of-occurrence and MD volumes, as both N_(BS) and δN_(BS)are first determined in a calibrating process. Thefrequency-of-occurence measurements are then comparedself-consisitently, by well known computational means, to a generalprobability distribution. This comprises a linear superposition of aPoisson function for free label and for analyte wherein the averageanalyte has bound N_(BS) labels. More specifically, the measureddistribution is fit to

    P(L).sub.fit =P.sub.F (n.sub.F, n.sub.F)+N.sub.BS P.sub.An (n.sub.An, n.sub.An)                                                 (25)

Subtraction of the Poisson formula P_(F) thereby results indetermination of P_(An). The value of n_(An) is obtained from P subbound, and in combination with V_(MD) for the range of MD volumes used,yields the desired analyte concentration ρ_(An).

This process is further illustrated by the case wherein the analytebiological entity is a macromolecule with three distinct,non-overlapping epitopes for which three non-cross reacting antibodymolecules are assumed. It is preferred to supply each of the threeantibody molecules at a concentration approximately equal to ten timesthe largest expected analyte concentration. Thus, if the largestexpected analyte concentration is about ρ_(An) =10⁻⁹ Molar, then each ofthe three antibodies is provided at a concentration of about 10⁻⁸ Molar.Following addition of the three antibodies to the sample, the resultingpreparation is mixed, and an incubation time of about 10 seconds to 3hours, preferably about 2 minutes to 20 minutes, is utilized. Thisallows diffusional encounter and an opportunity for the antibodies tobind to the analyte. Following this specific binding incubation, anyreagents or gelable material, if desired, are added. The resultingpreparation is mixed, and MDs are formed by any of the several methodsdescribed elsewhere in this disclosure, but preferably by dispersioninto a non-aqueous fluid. Generally, it is preferred to measure smallMDs, for which the probability difference for specifically bound labeland randomly distributed label is largest. That is, the difference

    ΔP=P.sub.An (n.sub.An,n.sub.An)-P.sub.F (n.sub.F,n.sub.F)(26)

is the probablistic basis for measuring analyte through the highlyimprobable association of N_(BS) labels within a MD compared to theprobability of random occurrence.

Although it is preferred to carry out the process of this invention byusing equilibrium conditions, or close to equilibrium conditionsfollowing exposure of analyte to LSBMs, it is also possible to utilizeconditions far from equilibrium. In such non-equilibrium cases, a samplecontaining analyte can be exposed to LSBMs for a significantly shortertime than needed for equilibrium, or for being close to equilibrium, andfewer completed complexes of analyte and N_(BS) LSBMs are formed. Thatis, not only are fewer complexes with exactly N_(BS) LSBMs formed, thereare more incomplete complexes formed wherein less than N_(BS) LSBMs arebound to each analyte entity. In spite of these less desirableattributes, a non-equilibrium measurement can be accomplished by thefurther step of making quantitative comparison, with otherwise the samenon-equilibrium conditions, to one or more calibrating measurementsemploying known concentrations of analyte.

The measurement process of this invention can be applied to very lowconcentrations of analyte molecules, as analyte molecules can beactually counted. In addition, the measurement can be made in solutionwithout the use of a solid phase which must be washed. Further, becausethe measurement process can involve a counting process, the inventionprovides means for measurements over a large dynamic range of analyteconcentrations, that is, from high concentrations to orders of magnitudelower concentrations.

Prior to the carrying out of the process of this invention, two or morelabeled specific binding molecules are obtained, using means well knownin the art, such that two or more labeled specific binding molecules areprepared, which are capable of binding to two or more binding sites onthe analyte. In the important case wherein LSBMs are antibodies, thisrequirement corresponds to using antibodies which bind to at least twonon-overlapping epitopes on the analyte, such that at least twoantibodies can be simultaneously and specifically bound to the analyte.Examples of such labeled specific binding molecules include (a)monoclonal antibodies with about one label molecule bound to eachantibody molecule, (b) antigen molecules with about one label moleculebound to each antigen entity, (c) monoclonal antibodies with about twolabel molecules of the same type are bound to each antibody molecule,(d) antigen molecules with about two label molecules of the same typebound to each antigen entity, and (e) polyclonal antibodies containingat least two antibodies capable of binding to at least twonon-overlapping eptiopes of the analyte entity.

Many different types of antlyte entities can be measured by the processof this invention. More specifically, analyte entities with at least twonon-overlapping and non-competing specific binding sites can bemeasured. In the important general class of analyte entities consistingof antigens for which the LSBMs are labeled antibodies, antigenicanalyte entities capable of independently binding antibodies at two ormore different sites can be measured. Examples of such analyte entitieswith two such sites include all antigens capable of assay by a sandwichassay, for example creatine kinase and hCG (human chorionicgonadotrophin). Examples of such analyte entities with three such sitesinclude proinsulin and the β-subunit of TSH (thyrotropin). In the caseof small molecules such as haptens, the assayed analyte entity with twoor more specific binding sites may consist of a hapten-carrier moleculecomplex.

Alternatively, if an analyte entity has multiple occurrence of one ormore binding sites, LSBMs with the same or different labels can be usedto multiply bind to each analyte entity. For example, an antigenicpolymer may have one or more multiple eptitopes, such that the sameantibody can specifically bind at multiple sites on the polymer, so thatsuch an antibody can be used in the process of this invention.Continuing this illustration, one or more labeled specific bindingmolecules in the form of one or more labeled antibody molecules areexposed to the analyte solution or sample, such that the antibodymolecules can bind at two or more repeated specific binding sites on thepolymer, thereby associating two or more labeled specific bindingmolecules with each analyte molecule. Other analytes with repeatedbinding sites include cells and viruses.

In the general practice of this invention, analyte entities such ascells, organelles, viruses, nucleic acids, antibodies, enzymes,structural proteins, hormones and drugs can be measured First, a samplecontaining such analyte entities is converted into a liquid solution orliquid suspension by any of the standard, well known means for preparinganalyte samples. The resulting analyte preparation is then exposed tolabeled specific binding molecules, such that there is a highprobability, following mixing and waiting for diffusion to occur, thatat least two LSBMs can bind to each analyte entity, thereby forminganalyte entity-complexes which contain at least two LSBMs. Second, someor all of this-reacted preparation can then be used to form MDs, suchthat there is a high probability that at least one MD, and preferrablyat least 10³ MDs, is individually occupied by an analyte entity-LSBMcomplex. Third, measurement means capable of measuring at least one LSBMis preferably utilized to measure MDs, and also the volumes of the MDs,such that by using statistical analysis, such as Poisson statistics theoccupation by LSBMs, and the associated volumes of the MDs, can bedetermined. Computation can then be used to compare the measuredfrequency-of-occurrence of LSBMS for LSBMs occuring singly and for LSBMsoccuring multiply, such that the excess of multiple occurances abovethat due to random occurrence is attributed to the specific binding ofLSBMs to analyte entities. The number of such above-random occurrencesis then used with statistical analysis to compute the number of analyteentities with bound LSBMs. This is combined with the volume of analyzedMDs to yield the number of analyte entities per volume in thepreparation from which MDs were formed, which number per volume is theconcentration of the analyte entities.

In another version of this process, it is only required that measurementmeans be capable of measuring N_(BS) labels, as this measurementsufficies if the occurrence of free label within MDs obeys n_(F)<N_(BS).

Labels for LSBMs suitable for use with this invention include enzymeactivity, biological activity and fluorescence. Fluorescence can bereadily measured when large numbers of fluorescent molecules arepresent, but it becomes increasingly more difficult for smaller numbers.Thus, the present detection limit is about 10³ molecules for fluoresceinmeasured in a flow cytometer, but still lower for measurement apparatussuch as quantitative fluorescence microscopy. A fundamental limitappears to relate to the number of fluorescence emission photons emittedbefore photodamage occurs, but it may be possible to measure individualfluorescent molecules such as phycoerythrin (see Mathies et al inFluorescence in the Biomedical Sciences, Liss, pp. 129-140, 1986). Thus,measurement based on singly or multiply labeled specific bindingreagents wherein an analyte molecule specifically binds several labeledspecific binding molecules is feasible. For example, by attaching anaverage of three fluorescent labels to each of three differentantibodies, nine fluorescent label molecules become associated with eachreacted analyte molecule. These can be readily measured by methodsdirected towards measurement of individual fluorescent molecules.

In general, however, the magitude of fluorescence signal associated withmeasurement of a fluorescence label is much too small. For this reason,the preferred embodiment of this invention involves the use of an activelabel, such as analyte activity related to vesicles, phage orbiochemical activity. It is preferred to utilize one or more activeenzymes, such that at least one enzyme label is measured through the useof optical measurements such as light scattering, light absorbance orcolorimetric, fluorescence, time-delayed fluorescence, phosphorescenceand chemiluminescence, but particularly fluorescence In much of thefollowing description the invention is described in terms of an enzymelabel, wherein the activity of at least one type of enzyme moleculeprovides the basis of measurement of the label, and is generally wellknown for use in macroscopic or non-microdroplet cases Such enzymeactivity measurement is accomplished generally by accumulatingfluorescent product of one or more enzyme catalyzed reactions within aMD containing the enzyme, and generally requires an incubation periodduring which the fluorescent product or fluorescent products canaccumulate It is preferred to provide conditions for a kinetic analysis,wherein substrates, co-factors, etc. are provided under conditions thatwill allow the enzyme catalyzed reaction to proceed at the maximumreaction velocity, which is at a rate equal to the turnover number forthe enzyme. Under these conditions, product accumulation occurs linearlywith time until product inhibition, substrate depletion, or otherwell-known enzyme reaction effects occur. Generally it is preferred toutilize measurements of MDs which have a high probability of beingunoccupied or individually occupied by analyte entities, that is,containing less than two analyte entities.

In addition to utilizing enzyme activity measurement directly, whereinone or more enzyme labels are measured by measuring one or morereactants of the corresponding enzyme catalyzed reactions, largersignals can generally be obtained by using enzyme channeling and/orenzyme cycling, both of which methods are well established for bulksolution use but have not previously been suggested or demonstrated foruse in MDs to measure individual, or small numbers of, enzyme molecules.Enzyme channeling, or use of linked enzyme reactions, consists ofproviding additional types of enzymes, such that a product of a firstenzyme catalyzed reaction serves as a substrate for a second enzymecatalyzed reaction, and a product of the second enzyme catalyzedreaction serves as a substrate for a third, and so on. Likewise, thewell established method of enzyme cycling provides amplification formeasuring enzyme labels by utilizing a cyclic reaction process whereinthe product of a first enzyme catalyzed reaction is a substrate orcofactor for a second enzyme catalyzed reaction, and a product of thesecond reaction is in turn a substrate for the first (see, for example,Siddle in Alternative Immunoassays, Collins (Ed.), Wiley, 1985). Ineither case, larger amounts of reaction product can be obtained within aMD. In order to utilize this version of the invention, additionalenzymes, substrates, cofactors and the like are provided in the sample,if not already present, so as to ensure that these additional enzymereactions reactants and enzyme will be present in each MD.

A sample containing particular analyte molecules whose analysis isdesired, can be converted into an aqueous solution or suspension by anyof a number of well known means, such as tissue homogenization,stirring, dissolution, and the like.

During or after the above treatment which yields a liquid solution orliquid suspension form of the sample, labeled specific binding moleculeswith enzyme labels are added to, and thoroughly mixed with the sample.The enzyme labeled specific binding molecules are provided in sufficientquantity, based on the expected maximum amount of the analyte molecules,so that essentially all of the analyte entitys will have reacted withand thereby bound the enzyme labeled specific binding molecules withinabout 10 seconds to 3 hours, and, under more optimal conditions, about 2minutes to 20 minutes.

Although substrates and/or cofactors for the enzymes catalyzed reactionscan be added in prior steps, it is preferable to add these reactantsjust prior to creation of liquid or gel microdroplets, in order tominimize the amounts of enzyme products are formed which would bedistributed into essentially all of the liquid or gel microdroplets, andwhich would result in an undesireable high background fluorescencesignal in all liquid or gel microdroplets.

A variety of methods can be used to measure the volumes of the MDs withand without LSBMs. The generation of some background, that is,fluorescence present uniformly in essentially all liquid or gelmicrodroplets, is sometimes desirable, as such background provides ameans of measuring the volume of microdroplets. In this case, all MDshave a low level of fluorescence, while MDs containing LSBMs, eitherunreacted or reacted so as to have bound with analyte analyte entities,have increased fluorescence. This is associated with the enzyme labelcatalyzing a reaction which has one or more fluorescent products, or iscoupled to one or more reactions which increase fluorescence.

In much of the measurement process it is possible to measure individualMDs, or to measure groups of MDs, with the latter often preferable ifthe sample concentration of analyte entity is low, as in that caserelatively few MDs are occupied by analyte entities. In the followingsection the quantity V_(group) refers to the total volume of a group ofMDs which are measured together, and it is understood that a group ofMDs can contain a number of MDs which is in the range 1 to 100 MDs,preferably 1 to 10 MDs.

As an alternative to utilizing background fluorescence from one or moreenzyme reactions for detection of, and measurement of V_(group) for eachanalyzed MD group, one or more fluorescent molecules can also beprovided at low concentration in the solution or suspension prior tocreation of MDs. This background concentration is selected tocorrespond, in the largest MD groups, to an amount which can bedistinguished from the fluorescence produced by one or two enzymemolecules within the volume of the largest MD groups used in theanalysis.

As another alternative, a fluorescent molecule type with fluorescenceproperties distinct from those used in enzyme assays within MD groupscan be used to provide detection of, and measurement of V_(group) ofeach MD group. This is useful for subsequent mathematical processing ofthe measurement data from a number of individual MD groups.

The aqueous solution/suspension form of the sample is then used tocreate LMDs or GMDs by any of several methods described elsewhere inthis disclosure. A preferred method is to add agarose, any tracerentities, reagents for enzyme assays, and the like, and to disperse theresulting solution/suspension in mineral oil or silicone fluid.

The resulting MD preparation is then incubated to allow the enzymecatalyzed reaction(s) to proceed, such that fluorescent product(s)accumulate preferentially in those MDs which contain enzyme labeledspecific binding molecules. Depending on the capabilities of the opticalmeasurement apparatus, and on the turnover number of the enzyme(s) used,the incubation time can range from about 30 minutes, or less, up toseveral hours.

There are a variety of choices of enzymes and measurement apparatusattributes. However, all are subject to the condition that eitherindividual enzyme molecule activity is measured, or that a sufficientlylarge number of enzymes are bound to individual analyte entities byenzyme labeled specific binding molecules that only this largeer numberis measured within each analyte occupied MD group. Desirable propertiesof an enzyme include high turnover number, stability, and specificity.

Following an incubation period some or all of the MD groups are measuredoptically, preferably using in an appratus with quantitative imageanalysis capability. Representative suitable apparatus includes flowcytometry apparatus, flow-through-microfluorimetry apparatus, opticalparticle analyzers apparatus, fluorescence microscopy apparatus, lightmicroscopy apparatus, image analysis apparatus and video recordingapparatus.

An important aspect of detection and measurement of one or moreindividual enzyme molecules was not explicitly mentioned or discussed inthe prior art (see Rotman, PNAS 47: 1981-1991, 1961). Specifially, ithas not been disclosed that it is important to use conditions whereinthe spontaneous rate of fluorescence production is sufficiently smallthat the catalytic effect of a single enzyme molecule in a small volumecan be distinguished against the background, generally increasing withtime, due to the spontaneous rate of fluorescence production. Such aspontaneous rate generally occurs for fluorgenic substrates, wherein thenon-fluorescent fluorogenic substrate spontaneously decays, andgenerates the same fluorescent molecules as the enzyme catalyzedreaction. Methods for avoiding singificant spontaneous fluorescence havenot been generally appreciated, or even explicitly acknowledged.Therefore, suitable methods for minimizing spontaneous fluorescence aredescribed as part of the present invention.

One general method is to determine, and then utilize, enzyme catalyzedreactions for which the fluorogenic substrates under the conditions ofthe assay yield fluorescence sufficiently small so that the fluorescencedue the product molecules catalyzed by one enzyme molecule isdetectable. In this approach, it is fundamental to consider the volume,V_(group), which contains the enzyme labeled specific binding molecule.The spontaneous rate of fluoresence accumulation is proportional toV_(group), while the enzyme catalyzed rate is, to a good approximation,independent of V_(group).

It is preferred to use a quantitative fluorescence microscope with imageanalysis capability wherein the amount of fluorescence emission fromindividual MDs, or groups of MDs, can be automatically measured.Alternatively, a flow cytometer with capability of measuringfluorescence at low levels can be used, such that the fluorescenceassociated with the accumulated product in a MD group due to one enzymemolecule can be detected, and distinguished from backgroundfluorescence. Optical measurement apparatus which allows detection of,for example, 10⁴ to 10⁵ fluorescein molecules is well known (see, forexample, Shapiro, Practical Flow Cytometry, Liss, New York, 1985).

It is preferred to use an enzyme such as β-galactosidase, with thefluorgenic substrate fluorescein-di-b-D-galactopyranoside, fluoresceinis the fluorescent product, has a large quantum yield, and is readilydetected and measured at the level of 10⁴ to 10⁵ fluorescein molecules(see, for example, Shapiro Practical Flow Cytometry, A. R. Liss, NewYork, 1985). Another useful substrate is FITC-diacetate (fluoresceinisothiocyanate-diacetate), which has the further desirable property ofbinding strongly and non-specifically to proteins. Thus, by providing asuitable amount of inexpensive protein such as BSA (bovine serumalbumin) mixed into the sample before GMDs are created, and subsequentlycoating the GMDs with a gel or other coating sufficient to retain BSA, asignificant fraction of the FITC is intercepted and bound by theprotein, so that an enzyme occupied GMD accumulates detectable GF.

Another useful enzyme catalyzed reaction is alkaline phosphataseutilized with the fluorgenic substrate 4-methylumbelliferyl phosphate,which catalyzes the degredation of this substrate into the fluorescentproduct 4-methyl umbelliferone plus the non-fluorescent productinorganic phosphate (see, for example, Guilbault Handbook of EnzymaticMethods of Analysis, Marcel Dekker, New York, 1976).

Following measurement of the amount of fluorescence(s) and volume,V_(group), of each MD group in a preparation, the resulting data isanalyzed in the following way, preferably using a computer. Theindividual values of V_(group) are summed, thereby providing adetermination of the sample actually analyzed. The amount offluorescence(s) in each MD group is determined, with respect topreviously carried out standard calibrations, so that the number ofenzyme molecules in each range of volumes of MDs is determined.Typically, the MDs have volume which range from about 5×10⁻¹⁰ to about5×10⁻⁷ ml, so that an analysis MDs of in the size (volume) ranges oftypically 5×10⁻¹⁰ to 7.9×10⁻¹⁰ ml, 8×10⁻¹⁰ to 1.19×10⁻⁹ ml, 1.2×10⁻⁹ to1.59×10⁻⁹ ml, 1.6×10⁻¹⁰ to 2×10⁻⁹ ml, and so on, is carried outmathematically. Such an analysis first makes use of the individualV_(group) determinations, and divides the MD group volume range intovolume intervals such that there are a significant number of MD groupsin each interval. Typically, MDs created by dispersion in mineral oilhave a volume distribution which rises sharply and then falls moderatelywith increasing V_(MD), with the consequence that most MD groups havevolumes just above an approximate cutoff size (e.g. 10 micron diameter,V_(group) ≈5×10⁻¹⁰ ml), and that it is useful to select volume intervalsof approximately a half order of magnitude in V_(group), as indicatedabove.

The Poisson distribution, is then used with iterative computations, tofind the best selfconsistent fit to each of the volume ranges. In thepresent case, if the concentration, or number density, of particularmolecules is ρ, then the average number of such molecules in MD groupsof volume V_(group) is n=ρV_(group). The Poisson distribution is used todescribe the statistical distribution of small entities such as cells ormolecules, such that the probability of finding n cells or molecules ina volume V_(group) is given by P (n,n). For purposes of interpretingmeasurements on a number of MD groups, n is the number of enzymemolecules found in each MD group, and n is the subsequently computedaverage number of enzyme molecules for each value of V_(group)Measurements of MD groups without enzyme molecules (n=0) and MD groupwith one enzyme molecule (n=1) are used with the Poisson distributionequation to predict the statistical frequency with which multiple enzymeoccupation of MD groups will occur due to random occupation. That is,the number of randomly occuring cases of n= 2, n=3, n=4, etc. arecomputed.

The number of MD groups found by measurement of fluorescence ofindividual MD groups to contain n=2, n=3, etc. enzyme labeled specificbinding molecules is then used to compute a best value of n for eachrange (interval) of V_(group) values, and then to compute the best valueof ρ by applying the equation ##EQU18## which is the intermediate of theassay, thereby being the number of analyte entities per volume in thediluted sample. It is straightforward to then compute a correctionfactor, f_(D), ##EQU19## for the dilution during the MD creationprocess, and thereby, to obtain the concentration, ρ_(analyte), ofanalyte entity in the (original) sample.

In this way, the process of this invention provides a determination ofthe amount of analyte in the original sample, and is based on thedetermination of individual analyte entities within individual MDgroups, as revealed by measurement of individual enzyme moleculeactivity. In order to obtain increased fluorescence it is generallypreferred to provide at least one incubation, wherein the enzymecatalyzed reactions which result in altered fluorescence, usuallyincreased fluorescence, can proceed.

Finally, in addition to providing and utilizing LSBMs, it is alsopossible to provide and utilize one or more types of interveningmolecules, such that the intervening molecules bind to one or more siteson the analyte entity, and one or more LSBMs are subsequently used. Forexample, a polyclonal preparation of unlabeled mouse antibodies can beused as intervening molecules, wherein these intervening molecules aremixed with the sample so as to allow binding of these unlabledintervening molecules to binding sites on the analyte entities. A goatanti-mouse antibody with an enzyme label, or other suitable label, canalso be added, such that the goat anti-mouse antibody is a LSBM.

It is often desirable to remove or purge the unbound interveningmolecules, which can be accomplished by adding beads with surface-boundand unlabeld goat anti-mouse antibodies. Such a purging step isgenerally useful, and consists of supplying purging entities such asbeads, non-biological particles, crystals, non-aqueous fluid inclusions,viable cells, dead cells, inactive cells, virus, spores, protoplasts andvesicles, which have surfaces with tightly bound molecules capable oftightly binding labeled specific binding molecules, which will remove,but not necessarily all, of the unbound intervening molecules, so as togreatly reduce the occurrence of complexes of intervening molecule-LSBMwhich are not associated with the analyte entities.

One or more purging steps can also be used in the case that only LSBMsare used, so as to bind much, but not necessarily all, of the free orunbound LSBMs which remains after the sample containing analyte entitieshas been exposed to the LSBMs, but before MDs are formed.

Significantly, it is not generally necessary to remove the purgingentites from the sample before forming MDs and carrying out theremainder of the process. In either the case of removing LSBM, whereinmostly free or unbound LSBMs are removed, or in the case in whichunlabled intervening molecules are removed, the association of theremoved LSBMs and/or intervening molecules with the purging entityprovides a general means for identifying such purging entities duringthe measurement process. General means for distinguishing the purgingentities includes measurements based on optical properties, mass densityproperties, acoustic properties, magnetic properties, electricalproperties and thermal properties, and it is preferred to use lightscattering, light absorbance or colorimetric, fluorescence, time-delayedfluorescence, phosphorescence and chemiluminescence. Thus, MDs whichcontain purging entities can be readily distinguished from MDs withoutpurging entities, so that the analysis of measured MDs can utilizemeasurements only from MDs without purging entities as the basis forcarrying out the desired assays and tests.

This invention will now be more specifically illustrated in thefollowing non-limiting examples.

EXAMPLE I Delivery of Water-Soluble Reagent to GMDs Surrounded byMineral Oil

GMDs were formed in a non-aqueous fluid by first preparing a liquidgellation medium according to the following steps:

1) 1 ml of aqueous buffer (200 mM acetate buffer, pH 8) was measuredout,

2) 50 mg agarose (Type VII, low melting temperature, Sigma Chemical) wasadded,

3) the preparation was heated to 80° C. (above the liquefactiontemperature)

4) the preparation was cooled to 40° C. (still above the gelationtemperature).

The enzyme alkaline phosphatase, was then added to the liquid gelationmedia, 10 ml of mineral oil was added, and vortexing used to produceliquid microdroplets (10-100 micron) which were gelled by cooling toabout 10° C., thereby incorporating enzyme into agarose GMDs which weresuspended in a tube of mineral oil (equivalent to parafin oil, heavy;Fisher; Saybold 162 min). A substrate for the enzyme,4-methylumbelliferone phosphate, was then dissolved in a small amount ofnon-polar solvent (acetone or DMSO), which was then added to a secondtube of mineral oil. Upon gently mixing these two tubes of mineral oil,the enzyme substrate was able to enter into the GMDs surrounded bymineral oil, and the enzyme catalyzed reaction occurred, which yielded afluorescent product which was visible in an ultraviolet microscope aftera period of a few minutes to hours, depending on the amount of enzymeactivity incorporated into the GMDs. This demonstrates the delivery of awater soluble chemical (4-methylumbelliferone phosphate) into GMDssurrounded by mineral oil.

EXAMPLE II Delivery of a Water-Soluble Compound to GMDs Using ReverseMicells

Agarose GMDs suspended in mineral oil were formed using methods similarto those of Example I. A small amount of water containing soap and afluorescent dye were then added to a second tube of mineral oil, andthen vortexed to create reverse micelles. The contents of the two tubes,one a mineral oil suspension of agarose GMDs, the other an emulsion ofreverse micells in mineral oil, were then mixed, so as to cause thereverse micelles to collide with the GMDs. This allowed the fluorescentdye contained in the micelles to be delivered into the GMDs, as wasobserved using fluorescence microscopy.

EXAMPLE III Rapid Delivery of Chemicals from GMDs to GMDs in SiliconeFluid

Agarose GMDs were formed by dispersion in 50 centipoise silicone fluid(Dow Corning) from a solution containing 1000 microgram per ml of4-methylumbelliferone (Blue fluorescence emission; ultravioletexcitation) in 0.1M Tris buffer at pH 8.0, thereby yielding fluorescentGMDs surrounded by the non-aqueous fluid comprising 50 cp siliconefluid. A drop of silicone fluid containing these Blue fluorescent GMDswas placed on a hemocytometer, as was a similar drop containingnon-fluorescent GMDs made by the same method, but without the4-methylumbelliferone (non-fluorescent GMDs). The two drops were gentlymixed, and the resulting mixed GMD preparation observed using afluorescence microscope.

Initially the two types of GMDs were readily distinguished as Bluefluorescent and non-fluorescent, but within a few minutes the initiallynon-fluorescent GMDs gradually acquired significant Blue fluoresence,thereby indicating a fairly rapid delivery of 4-methylumbelliferone frompre-loaded GMDs to the initially non-fluorescent GMDs.

EXAMPLE IV Slow Delivery of Chemicals from GMDs to GMDs in Mineral Oil

As in Example III, agarose GMDs were formed by dispersion, but in thiscase using mineral oil (equivalent to parafin oil, heavy; Fisher;Saybold 162 min). As in Example III, the solution from which GMDs wereformed contained 1000 microgram per ml of 4-methylumbelliferone (Bluefluorescence emission; ultraviolet excitation) in 0.1M Tris buffer at pH8.0, and thereby yielded fluorescent GMDs surrounded by the non-aqueousfluid comprising mineral oil. In contrast to Example III, no acquisitionof Blue fluorescence by the non-fluorescent GMDs was observed over aperiod of several minutes. However, after remaining overnight at roomtemperature the originally non-fluorescent GMDs became weakly Bluefluorescent, thereby indicating a slow delivery of 4-methylumbelliferonefrom pre-loaded GMDs to the initially non-fluorescent GMDs.

Equivalents

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to the specificembodiment of the invention described herein. Such equivlents areintended to be encompassed by the following claims.

We claim:
 1. A method for detecting an interaction between anamphiphilic compound and at least one biological entity, comprising thesteps of:a) disposing a biological entity in an aqueous medium, whereinthe biological entity is selected from the group consisting ofmicroorganisms, cells, protoplasts, small multicellular organisms,nucleic acid molecules, antibodies, antigens and viruses; b) forming theaqueous medium into microdroplets which are suspended in a non-aqueousphase, wherein the biological entity is contained within at least one ofthe microdroplets; c) introducing the amphiphilic compound into thenon-aqueous phase, whereby the amphiphilic compound migrates from thenon-aqueous phase into the microdroplet containing the biological entityand is partitioned between the non-aqueous phase and the microdroplet,permitting the amphiphilic compound and the biological entity in themicrodroplet to interact; and d) detecting the interaction between theamphiphilic compound and the biological entity.
 2. A method according toclaim 1 comprising the steps of:a) dissolving the amphiphilic compoundin a non-aqueous fluid, wherein the amphiphilic compound is soluble ineach of the non-aqueous fluid, the non-aqueous phase and themicrodroplets; b) contacting the non-aqueous fluid with the non-aqueousphase in which the microdroplets are suspended to permit partitioning ofthe amphiphilic compound between the non-aqueous fluid, the non-aqueousphase and the microdroplets, whereby the amphiphilic compound and thebiological entity in the microdroplet interact; and c) detecting theinteraction between the amphiphilic compound and the biological entity.3. A method for detecting an interaction between a water-solublecompound and at least one biological entity, comprising the steps of:a)disposing a biological entity in an aqueous medium, wherein thebiological entity is selected from the group consisting ofmicroorganisms, cells, protoplasts, small multicellular organisms,nucleic acid molecules, antibodies, antigens and viruses; b) forming theaqueous medium into microdroplets which are suspended in a non-aqueousphase, wherein the biological entity is contained within at least one ofthe microdroplets; c) combining the non-aqueous phase and themicrodroplets with an emulsion having a continuous phase which ismiscible with the non-aqueous phase and a discontinuous phase which ismiscible with the microdroplets and which contains the water-solublecompound, whereby at least a portion of the discontinuous phase combineswith the microdroplet containing the biological entity, permitting thewater-soluble compound and the biological entity in the microdroplet tointeract; and d) detecting the interaction between the water-solublecompound and the biological entity.
 4. The method of claim 3 wherein themicrodroplets are aqueous gel microdroplets.
 5. The method of claim 3further comprising adding at least one measurable tracer compound to thediscontinuous phase of the emulsion.
 6. The method of claim 5 whereinthe amount of water-soluble compound is determined by measuring theamount of the tracer compound introduced into the microdroplet.
 7. Themethod of claim 5 wherein the measurable tracer compound is afluorescent compound.
 8. The method of claim 5 wherein the measurabletracer compound is selected from the group consisting of fluorescein,rhodamine, coumarin, lucifer yellow and phycoerythrins.
 9. The method ofclaim 3 wherein the water-soluble compound is a substrate or a co-factorfor an enzyme-catalyzed reaction.
 10. The method of claim 3 wherein thewater-soluble compound is selected from the group consisting offluorescein-di-β-D-galactopyranoside, resorufin-β-D-galactopyranoside,fluorescein diacetate, carboxy fluorescein diacetate, fluoresceinisothiocyanate diacetate, fluorescein digalactoside, 4-methylumbelliferone butyrate, 4-methyl umbelliferone phosphate, 3-μ-methylfluorescein phosphate, diacetyl-2,7-dichloro fluorescein, homovanillicacid, a mixture of homovanillic acid and rhodamine lead, nicotinamideadenine dinucleotide and resazurin.
 11. The method of claim 3 whereinthe discontinuous phase of the emulsion is electrically charged toenhance contact with the microdroplets.